Isolated human kinase proteins, nucleic acid molecules encoding human kinase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the kinase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the kinase peptides, and methods of identifying modulators of the kinase peptides.

FIELD OF THE INVENTION

[0001] The present invention is in the field of kinase proteins that arerelated to the serine/threonine kinase subfamily, recombinant DNAmolecules, and protein production. The present invention specificallyprovides novel peptides and proteins that effect protein phosphorylationand nucleic acid molecules encoding such peptide and protein molecules,all of which are useful in the development of human therapeutics anddiagnostic compositions and methods.

BACKGROUND OF THE INVENTION

[0002] Protein Kinases

[0003] Kinases regulate many different cell proliferation,differentiation, and signaling processes by adding phosphate groups toproteins. Uncontrolled signaling has been implicated in a variety ofdisease conditions including inflammation, cancer, arteriosclerosis, andpsoriasis. Reversible protein phosphorylation is the main strategy forcontrolling activities of eukaryotic cells. It is estimated that morethan 1000 of the 10,000 proteins active in a typical mammalian cell arephosphorylated. The high-energy phosphate, which drives activation, isgenerally transferred from adenosine triphosphate molecules (ATP) to aparticular protein by protein kinases and removed from that protein byprotein phosphatases. Phosphorylation occurs in response toextracellular signals (hormones, neurotransmitters, growth anddifferentiation factors, etc), cell cycle checkpoints, and environmentalor nutritional stresses and is roughly analogous to turning on amolecular switch. When the switch goes on, the appropriate proteinkinase activates a metabolic enzyme, regulatory protein, receptor,cytoskeletal protein, ion channel or pump, or transcription factor.

[0004] The kinases comprise the largest known protein group, asuperfamily of enzymes with widely varied functions and specificities.They are usually named after their substrate, their regulatorymolecules, or some aspect of a mutant phenotype. With regard tosubstrates, the protein kinases may be roughly divided into two groups;those that phosphorylate tyrosine residues (protein tyrosine kinases,PTK) and those that phosphorylate serine or threonine residues(serine/threonine kinases, STK). A few protein kinases have dualspecificity and phosphorylate threonine and tyrosine residues. Almostall kinases contain a similar 250-300 amino acid catalytic domain. TheN-terminal domain, which contains subdomains I-IV, generally folds intoa two-lobed structure, which binds and orients the ATP (or GTP) donormolecule. The larger C terminal lobe, which contains subdomains VI A-XI,binds the protein substrate and carries out the transfer of the gammaphosphate from ATP to the hydroxyl group of a serine, threonine, ortyrosine residue. Subdomain V spans the two lobes.

[0005] The kinases may be categorized into families by the differentamino acid sequences (generally between 5 and 100 residues) located oneither side of, or inserted into loops of, the kinase domain. Theseadded amino acid sequences allow the regulation of each kinase as itrecognizes and interacts with its target protein. The primary structureof the kinase domains is conserved and can be further subdivided into 11subdomains. Each of the 11 subdomains contains specific residues andmotifs or patterns of amino acids that are characteristic of thatsubdomain and are highly conserved (Hardie, G. and Hanks, S. (1995) TheProtein Kinase Facts Books, Vol I:7-20 Academic Press, San Diego,Calif.).

[0006] The second messenger dependent protein kinases primarily mediatethe effects of second messengers such as cyclic AMP (cAMP), cyclic GMP,inositol triphosphate, phosphatidylinositol, 3,4,5-triphosphate,cyclic-ADPribose, arachidonic acid, diacylglycerol andcalcium-calmodulin. The cyclic-AMP dependent protein kinases (PKA) areimportant members of the STK family. Cyclic-AMP is an intracellularmediator of hormone action in all prokaryotic and animal cells that havebeen studied. Such hormone-induced cellular responses include thyroidhormone secretion, cortisol secretion, progesterone secretion, glycogenbreakdown, bone resorption, and regulation of heart rate and force ofheart muscle contraction. PKA is found in all animal cells and isthought to account for the effects of cyclic-AMP in most of these cells.Altered PKA expression is implicated in a variety of disorders anddiseases including cancer, thyroid disorders, diabetes, atherosclerosis,and cardiovascular disease (Isselbacher, K. J. et al. (1994) Harrison'sPrinciples of Internal Medicine, McGraw-Hill, New York, N.Y., pp.416-431, 1887).

[0007] Calcium-calmodulin (CaM) dependent protein kinases are alsomembers of STK family. Calmodulin is a calcium receptor that mediatesmany calcium regulated processes by binding to target proteins inresponse to the binding of calcium. The principle target protein inthese processes is CaM dependent protein kinases. CaM-kinases areinvolved in regulation of smooth muscle contraction (MLC kinase),glycogen breakdown (phosphorylase kinase), and neurotransmission (CaMkinase I and CaM kinase II). CaM kinase I phosphorylates a variety ofsubstrates including the neurotransmitter related proteins synapsin Iand II, the gene transcription regulator, CREB, and the cystic fibrosisconductance regulator protein, CFTR (Haribabu, B. et al. (1995) EMBOJournal 14:3679-86). CaM II kinase also phosphorylates synapsin atdifferent sites, and controls the synthesis of catecholamines in thebrain through phosphorylation and activation of tyrosine hydroxylase.Many of the CaM kinases are activated by phosphorylation in addition tobinding to CaM. The kinase may autophosphorylate itself, or bephosphorylated by another kinase as part of a “kinase cascade”.

[0008] Another ligand-activated protein kinase is 5′-AMP-activatedprotein kinase (AMPK) (Gao, G. et al. (1996) J. Biol Chem. 15:8675-81).Mammalian AMPK is a regulator of fatty acid and sterol synthesis throughphosphorylation of the enzymes acetyl-CoA carboxylase andhydroxymethylglutaryl-CoA reductase and mediates responses of thesepathways to cellular stresses such as heat shock and depletion ofglucose and ATP. AMPK is a heterotrimeric complex comprised of acatalytic alpha subunit and two non-catalytic beta and gamma subunitsthat are believed to regulate the activity of the alpha subunit.Subunits of AMPK have a much wider distribution in non-lipogenic tissuessuch as brain, heart, spleen, and lung than expected. This distributionsuggests that its role may extend beyond regulation of lipid metabolismalone.

[0009] The mitogen-activated protein kinases (MAP) are also members ofthe STK family. MAP kinases also regulate intracellular signalingpathways. They mediate signal transduction from the cell surface to thenucleus via phosphorylation cascades. Several subgroups have beenidentified, and each manifests different substrate specificities andresponds to distinct extracellular stimuli (Egan, S. E. and Weinberg, R.A. (1993) Nature 365:781-783). MAP kinase signaling pathways are presentin mammalian cells as well as in yeast. The extracellular stimuli thatactivate mammalian pathways include epidermal growth factor (EGF),ultraviolet light, hyperosmolar medium, heat shock, endotoxiclipopolysaccharide (LPS), and pro-inflammatory cytokines such as tumornecrosis factor (TNF) and interleukin-1 (IL-1).

[0010] PRK (proliferation-related kinase) is a serum/cytokine inducibleSTK that is involved in regulation of the cell cycle and cellproliferation in human megakaroytic cells (Li, B. et al. (1996) J. Biol.Chem. 271:19402-8). PRK is related to the polo (derived from humans pologene) family of STKs implicated in cell division. PRK is downregulatedin lung tumor tissue and may be a proto-oncogene whose deregulatedexpression in normal tissue leads to oncogenic transformation. AlteredMAP kinase expression is implicated in a variety of disease conditionsincluding cancer, inflammation, immune disorders, and disordersaffecting growth and development.

[0011] The cyclin-dependent protein kinases (CDKs) are another group ofSTKs that control the progression of cells through the cell cycle.Cyclins are small regulatory proteins that act by binding to andactivating CDKs that then trigger various phases of the cell cycle byphosphorylating and activating selected proteins involved in the mitoticprocess. CDKs are unique in that they require multiple inputs to becomeactivated. In addition to the binding of cyclin, CDK activation requiresthe phosphorylation of a specific threonine residue and thedephosphorylation of a specific tyrosine residue.

[0012] Protein tyrosine kinases, PTKs, specifically phosphorylatetyrosine residues on their target proteins and may be divided intotransmembrane, receptor PTKs and nontransmembrane, non-receptor PTKs.Transmembrane protein-tyrosine kinases are receptors for most growthfactors. Binding of growth factor to the receptor activates the transferof a phosphate group from ATP to selected tyrosine side chains of thereceptor and other specific proteins. Growth factors (GF) associatedwith receptor PTKs include; epidermal GF, platelet-derived GF,fibroblast GF, hepatocyte GF, insulin and insulin-like GFs, nerve GF,vascular endothelial GF, and macrophage colony stimulating factor.

[0013] Non-receptor PTKs lack transmembrane regions and, instead, formcomplexes with the intracellular regions of cell surface receptors. Suchreceptors that function through non-receptor PTKs include those forcytokines, hormones (growth hormone and prolactin) and antigen-specificreceptors on T and B lymphocytes.

[0014] Many of these PTKs were first identified as the products ofmutant oncogenes in cancer cells where their activation was no longersubject to normal cellular controls. In fact, about one third of theknown oncogenes encode PTKs, and it is well known that cellulartransformation (oncogenesis) is often accompanied by increased tyrosinephosphorylation activity (Carbonneau H and Tonks NK (1992) Annu. Rev.Cell. Biol. 8:463-93). Regulation of PTK activity may therefore be animportant strategy in controlling some types of cancer.

[0015] Kinase proteins, particularly members of the serine/threoninekinase subfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of kinase proteins. The present invention advances the stateof the art by providing previously unidentified human kinase proteinsthat have homology to members of the serine/threonine kinase subfamily.

SUMMARY OF THE INVENTION

[0016] The present invention is based in part on the identification ofamino acid sequences of human kinase peptides and proteins that arerelated to the serine/threonine kinase subfamily, as well as allelicvariants and other mammalian orthologs thereof. These unique peptidesequences, and nucleic acid sequences that encode these peptides, can beused as models for the development of human therapeutic targets, aid inthe identification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate kinase activity incells and tissues that express the kinase. Experimental data as providedin FIG. 1 indicates expression in lung carcinoma and placenta.

DESCRIPTION OF THE FIGURE SHEETS

[0017]FIG. 1 provides the nucleotide sequence of a cDNA molecule ortranscript sequence that encodes the kinase protein of the presentinvention. (SEQ ID NO:1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. 1 indicates expression in lungcarcinoma and placenta.

[0018]FIG. 2 provides the predicted amino acid sequence of the kinase ofthe present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

[0019]FIG. 3 provides genomic sequences that span the gene encoding thekinase protein of the present invention. (SEQ ID NO:3) In additionstructure and functional information, such as intron/exon structure,promoter location, etc., is provided where available, allowing one toreadily determine specific uses of inventions based on this molecularsequence. As illustrated in FIG. 3, SNPs were identified at 33positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

[0020] The present invention is based on the sequencing of the humangenome. During the sequencing and assembly of the human genome, analysisof the sequence information revealed previously unidentified fragmentsof the human genome that encode peptides that share structural and/orsequence homology to protein/peptide/domains identified andcharacterized within the art as being a kinase protein or part of akinase protein and are related to the serine/threonine kinase subfamily.Utilizing these sequences, additional genomic sequences were assembledand transcript and/or cDNA sequences were isolated and characterized.Based on this analysis, the present invention provides amino acidsequences of human kinase peptides and proteins that are related to theserine/threonine kinase subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these kinase peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the kinase of the present invention.

[0021] In addition to being previously unknown, the peptides that areprovided in the present invention are selected based on their ability tobe used for the development of commercially important products andservices. Specifically, the present peptides are selected based onhomology and/or structural relatedness to known kinase proteins of theserine/threonine kinase subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in lungcarcinoma and placenta. The art has clearly established the commercialimportance of members of this family of proteins and proteins that haveexpression patterns similar to that of the present gene. Some of themore specific features of the peptides of the present invention, and theuses thereof, are described herein, particularly in the Background ofthe Invention and in the annotation provided in the Figures, and/or areknown within the art for each of the known serine/threonine kinasefamily or subfamily of kinase proteins.

Specific Embodiments

[0022] Peptide Molecules

[0023] The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thekinase family of proteins and are related to the serine/threonine kinasesubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the kinase peptides of the present invention, kinase peptides,or peptides/proteins of the present invention.

[0024] The present invention provides isolated peptide and proteinmolecules that consist of, consist essentially of, or comprise the aminoacid sequences of the kinase peptides disclosed in the FIG. 2, (encodedby the nucleic acid molecule shown in FIG. 1, transcript/cDNA or FIG. 3,genomic sequence), as well as all obvious variants of these peptidesthat are within the art to make and use. Some of these variants aredescribed in detail below.

[0025] As used herein, a peptide is said to be “isolated” or “purified”when it is substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

[0026] In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

[0027] The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thekinase peptide having less than about 30% (by dry weight) chemicalprecursors or other chemicals, less than about 20% chemical precursorsor other chemicals, less than about 10% chemical precursors or otherchemicals, or less than about 5% chemical precursors or other chemicals.

[0028] The isolated kinase peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inlung carcinoma and placenta. For example, a nucleic acid moleculeencoding the kinase peptide is cloned into an expression vector, theexpression vector introduced into a host cell and the protein expressedin the host cell. The protein can then be isolated from the cells by anappropriate purification scheme using standard protein purificationtechniques. Many of these techniques are described in detail below.

[0029] Accordingly, the present invention provides proteins that consistof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). The amino acid sequence of such a protein is providedin FIG. 2. A protein consists of an amino acid sequence when the aminoacid sequence is the final amino acid sequence of the protein.

[0030] The present invention further provides proteins that consistessentially of the amino acid sequences provided in FIG. 2 (SEQ IDNO:2), for example, proteins encoded by the transcript/cDNA nucleic acidsequences shown in FIG. 1 (SEQ ID NO:1) and the genomic sequencesprovided in FIG. 3 (SEQ ID NO:3). A protein consists essentially of anamino acid sequence when such an amino acid sequence is present withonly a few additional amino acid residues, for example from about 1 toabout 100 or so additional residues, typically from 1 to about 20additional residues in the final protein.

[0031] The present invention further provides proteins that comprise theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the kinase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

[0032] The kinase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a kinase peptide operativelylinked to a heterologous protein having an amino acid sequence notsubstantially homologous to the kinase peptide. “Operatively linked”indicates that the kinase peptide and the heterologous protein are fusedin-frame. The heterologous protein can be fused to the N-terminus orC-terminus of the kinase peptide.

[0033] In some uses, the fusion protein does not affect the activity ofthe kinase peptide per se. For example, the fusion protein can include,but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins,particularly poly-His fusions, can facilitate the purification ofrecombinant kinase peptide. In certain host cells (e.g., mammalian hostcells), expression and/or secretion of a protein can be increased byusing a heterologous signal sequence.

[0034] A chimeric or fusion protein can be produced by standardrecombinant DNA techniques. For example, DNA fragments coding for thedifferent protein sequences are ligated together in-frame in accordancewith conventional techniques. In another embodiment, the fusion gene canbe synthesized by conventional techniques including automated DNAsynthesizers. Alternatively, PCR amplification of gene fragments can becarried out using anchor primers which give rise to complementaryoverhangs between two consecutive gene fragments which can subsequentlybe annealed and re-amplified to generate a chimeric gene sequence (seeAusubel et al., Current Protocols in Molecular Biology, 1992). Moreover,many expression vectors are commercially available that already encode afusion moiety (e.g., a GST protein). A kinase peptide-encoding nucleicacid can be cloned into such an expression vector such that the fusionmoiety is linked in-frame to the kinase peptide.

[0035] As mentioned above, the present invention also provides andenables obvious variants of the amino acid sequence of the proteins ofthe present invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

[0036] Such variants can readily be identified/made using moleculartechniques and the sequence information disclosed herein. Further, suchvariants can readily be distinguished from other peptides based onsequence and/or structural homology to the kinase peptides of thepresent invention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

[0037] To determine the percent identity of two amino acid sequences ortwo nucleic acid sequences, the sequences are aligned for optimalcomparison purposes (e.g., gaps can be introduced in one or both of afirst and a second amino acid or nucleic acid sequence for optimalalignment and non-homologous sequences can be disregarded for comparisonpurposes). In a preferred embodiment, at least 30%, 40%, 50%, 60%, 70%,80%, or 90% or more of the length of a reference sequence is aligned forcomparison purposes. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein amino acid or nucleic acid “identity” is equivalent to aminoacid or nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

[0038] The comparison of sequences and determination of percent identityand similarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

[0039] The nucleic acid and protein sequences of the present inventioncan further be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

[0040] Full-length pre-processed forms, as well as mature processedforms, of proteins that comprise one of the peptides of the presentinvention can readily be identified as having complete sequence identityto one of the kinase peptides of the present invention as well as beingencoded by the same genetic locus as the kinase peptide provided herein.As indicated by the data presented in FIG. 3, the map position wasdetermined to be on chromosome 13 by ePCR, and confirmed with radiationhybrid mapping.

[0041] Allelic variants of a kinase peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the kinase peptide as well asbeing encoded by the same genetic locus as the kinase peptide providedherein. Genetic locus can readily be determined based on the genomicinformation provided in FIG. 3, such as the genomic sequence mapped tothe reference human. As indicated by the data presented in FIG. 3, themap position was determined to be on chromosome 13 by ePCR, andconfirmed with radiation hybrid mapping. As used herein, two proteins(or a region of the proteins) have significant homology when the aminoacid sequences are typically at least about 70-80%, 80-90%, and moretypically at least about 90-95% or more homologous. A significantlyhomologous amino acid sequence, according to the present invention, willbe encoded by a nucleic acid sequence that will hybridize to a kinasepeptide encoding nucleic acid molecule under stringent conditions asmore fully described below.

[0042]FIG. 3 provides information on SNPs that have been found in a geneencoding the kinase proteins of the present invention. Thirty-three SNPswere identified. The changes in the amino acid sequence that these SNPscause can readily be determined using the universal genetic code and theprotein sequence provided in FIG. 2 as a base.

[0043] Paralogs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide, as being encoded by a gene from humans,and as having similar activity or function. Two proteins will typicallybe considered paralogs when the amino acid sequences are typically atleast about 60% or greater, and more typically at least about 70% orgreater homology through a given region or domain. Such paralogs will beencoded by a nucleic acid sequence that will hybridize to a kinasepeptide encoding nucleic acid molecule under moderate to stringentconditions as more fully described below.

[0044] Orthologs of a kinase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the kinase peptide as well as being encoded by a gene fromanother organism. Preferred orthologs will be isolated from mammals,preferably primates, for the development of human therapeutic targetsand agents. Such orthologs will be encoded by a nucleic acid sequencethat will hybridize to a kinase peptide encoding nucleic acid moleculeunder moderate to stringent conditions, as more fully described below,depending on the degree of relatedness of the two organisms yielding theproteins.

[0045] Non-naturally occurring variants of the kinase peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the kinase peptide. Forexample, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a kinase peptide by another amino acid of like characteristics.Typically seen as conservative substitutions are the replacements, onefor another, among the aliphatic amino acids Ala, Val, Leu, and Ile;interchange of the hydroxyl residues Ser and Thr; exchange of the acidicresidues Asp and Glu; substitution between the amide residues Asn andGln; exchange of the basic residues Lys and Arg; and replacements amongthe aromatic residues Phe and Tyr. Guidance concerning which amino acidchanges are likely to be phenotypically silent are found in Bowie etal., Science 247:1306-1310 (1990).

[0046] Variant kinase peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in noncritical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

[0047] Non-functional variants typically contain one or morenon-conservative amino acid substitutions, deletions, insertions,inversions, or truncation or a substitution, insertion, inversion, ordeletion in a critical residue or critical region.

[0048] Amino acids that are essential for function can be identified bymethods known in the art, such as site-directed mutagenesis oralanine-scanning mutagenesis (Cunningham et al., Science 244:1081-1085(1989)), particularly using the results provided in FIG. 2. The latterprocedure introduces single alanine mutations at every residue in themolecule. The resulting mutant molecules are then tested for biologicalactivity such as kinase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

[0049] The present invention further provides fragments of the kinasepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

[0050] As used herein, a fragment comprises at least 8, 10, 12, 14, 16,or more contiguous amino acid residues from a kinase peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the kinase peptide or could be chosen forthe ability to perform a function, e.g. bind a substrate or act as animmunogen. Particularly important fragments are biologically activefragments, peptides that are, for example, about 8 or more amino acidsin length. Such fragments will typically comprise a domain or motif ofthe kinase peptide, e.g., active site, a transmembrane domain or asubstrate-binding domain. Further, possible fragments include, but arenot limited to, domain or motif containing fragments, soluble peptidefragments, and fragments containing immunogenic structures. Predicteddomains and functional sites are readily identifiable by computerprograms well known and readily available to those of skill in the art(e.g., PROSITE analysis). The results of one such analysis are providedin FIG. 2.

[0051] Polypeptides often contain amino acids other than the 20 aminoacids commonly referred to as the 20 naturally occurring amino acids.Further, many amino acids, including the terminal amino acids, may bemodified by natural processes, such as processing and otherpost-translational modifications, or by chemical modification techniqueswell known in the art. Common modifications that occur naturally inkinase peptides are described in basic texts, detailed monographs, andthe research literature, and they are well known to those of skill inthe art (some of these features are identified in FIG. 2).

[0052] Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

[0053] Such modifications are well known to those of skill in the artand have been described in great detail in the scientific literature.Several particularly common modifications, glycosylation, lipidattachment, sulfation, gamma-carboxylation of glutamic acid residues,hydroxylation and ADP-ribosylation, for instance, are described in mostbasic texts, such as Proteins—Structure and Molecular Properties, 2ndEd., T. E. Creighton, W. H. Freeman and Company, New York (1993). Manydetailed reviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

[0054] Accordingly, the kinase peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature kinase peptide is fused withanother compound, such as a compound to increase the half-life of thekinase peptide (for example, polyethylene glycol), or in which theadditional amino acids are fused to the mature kinase peptide, such as aleader or secretory sequence or a sequence for purification of themature kinase peptide or a pro-protein sequence.

[0055] Protein/Peptide Uses

[0056] The proteins of the present invention can be used in substantialand specific assays related to the functional information provided inthe Figures; to raise antibodies or to elicit another immune response;as a reagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in a kinase-effectorprotein interaction or kinase-ligand interaction), the protein can beused to identify the binding partner/ligand so as to develop a system toidentify inhibitors of the binding interaction. Any or all of these usesare capable of being developed into reagent grade or kit format forcommercialization as commercial products.

[0057] Methods for performing the uses listed above are well known tothose skilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

[0058] The potential uses of the peptides of the present invention arebased primarily on the source of the protein as well as the class/actionof the protein. For example, kinases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the kinase. Experimental data as providedin FIG. 1 indicates that kinase proteins of the present invention areexpressed in lung carcinoma, as indicated by virtual northern blotanalysis, and placenta, which is the cDNA library source from which thecDNA clone came from. A large percentage of pharmaceutical agents arebeing developed that modulate the activity of kinase proteins,particularly members of the serine/threonine kinase subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates expression in lungcarcinoma and placenta. Such uses can readily be determined using theinformation provided herein, that which is known in the art, and routineexperimentation.

[0059] The proteins of the present invention (including variants andfragments that may have been disclosed prior to the present invention)are useful for biological assays related to kinases that are related tomembers of the serine/threonine kinase subfamily. Such assays involveany of the known kinase functions or activities or properties useful fordiagnosis and treatment of kinase-related conditions that are specificfor the subfamily of kinases that the one of the present inventionbelongs to, particularly in cells and tissues that express the kinase.Experimental data as provided in FIG. 1 indicates that kinase proteinsof the present invention are expressed in lung carcinoma, as indicatedby virtual northern blot analysis, and placenta, which is the cDNAlibrary source from which the cDNA clone came from.

[0060] The proteins of the present invention are also useful in drugscreening assays, in cell-based or cell-free systems. Cell-based systemscan be native, i.e., cells that normally express the kinase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in lung carcinoma and placenta. In an alternateembodiment, cell-based assays involve recombinant host cells expressingthe kinase protein.

[0061] The polypeptides can be used to identify compounds that modulatekinase activity of the protein in its natural state or an altered formthat causes a specific disease or pathology associated with the kinase.Both the kinases of the present invention and appropriate variants andfragments can be used in high-throughput screens to assay candidatecompounds for the ability to bind to the kinase. These compounds can befurther screened against a functional kinase to determine the effect ofthe compound on the kinase activity. Further, these compounds can betested in animal or invertebrate systems to determineactivity/effectiveness. Compounds can be identified that activate(agonist) or inactivate (antagonist) the kinase to a desired degree.

[0062] Further, the proteins of the present invention can be used toscreen a compound for the ability to stimulate or inhibit interactionbetween the kinase protein and a molecule that normally interacts withthe kinase protein, e.g. a substrate or a component of the signalpathway that the kinase protein normally interacts (for example, anotherkinase). Such assays typically include the steps of combining the kinaseprotein with a candidate compound under conditions that allow the kinaseprotein, or fragment, to interact with the target molecule, and todetect the formation of a complex between the protein and the target orto detect the biochemical consequence of the interaction with the kinaseprotein and the target, such as any of the associated effects of signaltransduction such as protein phosphorylation, cAMP turnover, andadenylate cyclase activation, etc.

[0063] Candidate compounds include, for example, 1) peptides such assoluble peptides, including Ig-tailed fusion peptides and members ofrandom peptide libraries (see, e.g., Lam et al., Nature 354:82-84(1991); Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

[0064] One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantkinases or appropriate fragments containing mutations that affect kinasefunction and thus compete for substrate. Accordingly, a fragment thatcompetes for substrate, for example with a higher affinity, or afragment that binds substrate but does not allow release, is encompassedby the invention.

[0065] The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) kinase activity. Theassays-typically involve an assay of events in the signal transductionpathway that indicate kinase activity. Thus, the phosphorylation of asubstrate, activation of a protein, a change in the expression of genesthat are up- or down-regulated in response to the kinase proteindependent signal cascade can be assayed.

[0066] Any of the biological or biochemical functions mediated by thekinase can be used as an endpoint assay. These include all of thebiochemical or biochemical/biological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the kinase can be assayed.Experimental data as provided in FIG. 1 indicates that kinase proteinsof the present invention are expressed in lung carcinoma, as indicatedby virtual northern blot analysis, and placenta, which is the cDNAlibrary source from which the cDNA clone came from.

[0067] Binding and/or activating compounds can also be screened by usingchimeric kinase proteins in which the amino terminal extracellulardomain, or parts thereof, the entire transmembrane domain or subregions,such as any of the seven transmembrane segments or any of theintracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native kinase. Accordingly, a different set of signaltransduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the kinase is derived.

[0068] The proteins of the present invention are also useful incompetition binding assays in methods designed to discover compoundsthat interact with the kinase (e.g. binding partners and/or ligands).Thus, a compound is exposed to a kinase polypeptide under conditionsthat allow the compound to bind or to otherwise interact with thepolypeptide. Soluble kinase polypeptide is also added to the mixture. Ifthe test compound interacts with the soluble kinase polypeptide, itdecreases the amount of complex formed or activity from the kinasetarget. This type of assay is particularly useful in cases in whichcompounds are sought that interact with specific regions of the kinase.Thus, the soluble polypeptide that competes with the target kinaseregion is designed to contain peptide sequences corresponding to theregion of interest.

[0069] To perform cell free drug screening assays, it is sometimesdesirable to immobilize either the kinase protein, or fragment, or itstarget molecule to facilitate separation of complexes from uncomplexedforms of one or both of the proteins, as well as to accommodateautomation of the assay.

[0070] Techniques for immobilizing proteins on matrices can be used inthe drug screening assays. In one embodiment, a fusion protein can beprovided which adds a domain that allows the protein to be bound to amatrix. For example, glutathione-S-transferase fusion proteins can beadsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis,Mo.) or glutathione derivatized microtitre plates, which are thencombined with the cell lysates (e.g., ³⁵S-labeled) and the candidatecompound, and the mixture incubated under conditions conducive tocomplex formation (e.g., at physiological conditions for salt and pH).Following incubation, the beads are washed to remove any unbound label,and the matrix immobilized and radiolabel determined directly, or in thesupernatant after the complexes are dissociated. Alternatively, thecomplexes can be dissociated from the matrix, separated by SDS-PAGE, andthe level of kinase-binding protein found in the bead fractionquantitated from the gel using standard electrophoretic techniques. Forexample, either the polypeptide or its target molecule can beimmobilized utilizing conjugation of biotin and streptavidin usingtechniques well known in the art. Alternatively, antibodies reactivewith the protein but which do not interfere with binding of the proteinto its target molecule can be derivatized to the wells of the plate, andthe protein trapped in the wells by antibody conjugation. Preparationsof a kinase-binding protein and a candidate compound are incubated inthe kinase protein-presenting wells and the amount of complex trapped inthe well can be quantitated. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with thekinase protein target molecule, or which are reactive with kinaseprotein and compete with the target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with thetarget molecule.

[0071] Agents that modulate one of the kinases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

[0072] Modulators of kinase protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the kinase pathway, by treating cells or tissuesthat express the kinase. Experimental data as provided in FIG. 1indicates expression in lung carcinoma and placenta. These methods oftreatment include the steps of administering a modulator of kinaseactivity in a pharmaceutical composition to a subject in need of suchtreatment, the modulator being identified as described herein.

[0073] In yet another aspect of the invention, the kinase proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the kinase and are involved in kinase activity.Such kinase-binding proteins are also likely to be involved in thepropagation of signals by the kinase proteins or kinase targets as, forexample, downstream elements of a kinase-mediated signaling pathway.Alternatively, such kinase-binding proteins are likely to be kinaseinhibitors.

[0074] The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a kinase proteinis fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming akinase-dependent complex, the DNA-binding and activation domains of thetranscription factor are brought into close proximity. This proximityallows transcription of a reporter gene (e.g., LacZ) which is operablylinked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the kinase protein.

[0075] This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a kinase-modulating agent, an antisense kinasenucleic acid molecule, a kinase-specific antibody, or a kinase-bindingpartner) can be used in an animal or other model to determine theefficacy, toxicity, or side effects of treatment with such an agent.Alternatively, an agent identified as described herein can be used in ananimal or other model to determine the mechanism of action of such anagent. Furthermore, this invention pertains to uses of novel agentsidentified by the above-described screening assays for treatments asdescribed herein.

[0076] The kinase proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in lung carcinoma and placenta. The method involvescontacting a biological sample with a compound capable of interactingwith the kinase protein such that the interaction can be detected. Suchan assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0077] One agent for detecting a protein in a sample is an antibodycapable of selectively binding to protein. A biological sample includestissues, cells and biological fluids isolated from a subject, as well astissues, cells and fluids present within a subject.

[0078] The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution; deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered kinase activity in cell-based orcell-free assay, alteration in substrate or antibody-binding pattern,altered isoelectric point, direct amino acid sequencing, and any otherof the known assay techniques useful for detecting mutations in aprotein. Such an assay can be provided in a single detection format or amulti-detection format such as an antibody chip array.

[0079] In vitro techniques for detection of peptide include enzymelinked immunosorbent assays (ELISAs), Western blots,immunoprecipitations and immunofluorescence using a detection reagent,such as an antibody or protein binding agent. Alternatively, the peptidecan be detected in vivo in a subject by introducing into the subject alabeled anti-peptide antibody or other types of detection agent. Forexample; the antibody can be labeled with a radioactive marker whosepresence and location in a subject can be detected by standard imagingtechniques. Particularly useful are methods that detect the allelicvariant of a peptide expressed in a subject and methods which detectfragments of a peptide in a sample.

[0080] The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the kinase protein in which one ormore of the kinase functions in one population is different from thosein another population. The peptides thus allow a target to ascertain agenetic predisposition that can affect treatment modality. Thus, in aligand-based treatment, polymorphism may give rise to amino terminalextracellular domains and/or other substrate-binding regions that aremore or less active in substrate binding, and kinase activation.Accordingly, substrate dosage would necessarily be modified to maximizethe therapeutic effect within a given population containing apolymorphism. As an alternative to genotyping, specific polymorphicpeptides could be identified.

[0081] The peptides are also useful for treating a disordercharacterized by an absence of, inappropriate, or unwanted expression ofthe protein. Experimental data as provided in FIG. 1 indicatesexpression in lung carcinoma and placenta. Accordingly, methods fortreatment include the use of the kinase protein or fragments.

[0082] Antibodies

[0083] The invention also provides antibodies that selectively bind toone of the peptides of the present invention, a protein comprising sucha peptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

[0084] As used herein, an antibody is defined in terms consistent withthat recognized within the art: they are multi-subunit proteins producedby a mammalian organism in response to an antigen challenge. Theantibodies of the present invention include polyclonal antibodies andmonoclonal antibodies, as well as fragments of such antibodies,including, but not limited to, Fab or F(ab′)₂, and Fv fragments.

[0085] Many methods are known for generating and/or identifyingantibodies to a given target peptide. Several such methods are describedby Harlow, Antibodies, Cold Spring Harbor Press, (1989).

[0086] In general, to generate antibodies an isolated peptide is used asan immunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering functional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

[0087] Antibodies are preferably prepared from regions or discretefragments of the kinase proteins. Antibodies can be prepared from anyregion of the peptide as described herein. However, preferred regionswill include those involved in function/activity and/or kinase/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

[0088] An antigenic fragment will typically comprise at least 8contiguous amino acid residues. The antigenic peptide can comprise,however, at least 10, 12, 14, 16 or more amino acid residues. Suchfragments can be selected on a physical property, such as fragmentscorrespond to regions that are located on the surface of the protein,e.g., hydrophilic regions or can be selected based on sequenceuniqueness (see FIG. 2).

[0089] Detection on an antibody of the present invention can befacilitated by coupling (i.e., physically linking) the antibody to adetectable substance. Examples of detectable substances include variousenzymes, prosthetic groups, fluorescent materials, luminescentmaterials, bioluminescent materials, and radioactive materials. Examplesof suitable enzymes include horseradish peroxidase, alkalinephosphatase, β-galactosidase, or acetylcholinesterase; examples ofsuitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

[0090] Antibody Uses

[0091] The antibodies can be used to isolate one of the proteins of thepresent invention by standard techniques, such as affinitychromatography or immunoprecipitation. The antibodies can facilitate thepurification of the natural protein from cells and recombinantlyproduced protein expressed in host cells. In addition, such antibodiesare useful to detect the presence of one of the proteins of the presentinvention in cells or tissues to determine the pattern of expression ofthe protein among various tissues in an organism and over the course ofnormal development. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in lungcarcinoma, as indicated by virtual northern blot analysis, and placenta,which is the cDNA library source from which the cDNA clone came from.Further, such antibodies can be used to detect protein in situ, invitro, or in a cell lysate or supernatant in order to evaluate theabundance and pattern of expression. Also, such antibodies can be usedto assess abnormal tissue distribution or abnormal expression duringdevelopment or progression of a biological condition. Antibody detectionof circulating fragments of the full length protein can be used toidentify turnover.

[0092] Further, the antibodies can be used to assess expression indisease states such as in active stages of the disease or in anindividual with a predisposition toward disease related to the protein'sfunction. When a disorder is caused by an inappropriate tissuedistribution, developmental expression, level of expression of theprotein, or expressed/processed form, the antibody can be preparedagainst the normal protein. Experimental data as provided in FIG. 1indicates expression in lung carcinoma and placenta. If a disorder ischaracterized by a specific mutation in the protein, antibodies specificfor this mutant protein can be used to assay for the presence of thespecific mutant protein.

[0093] The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in lungcarcinoma and placenta. The diagnostic uses can be applied, not only ingenetic testing, but also in monitoring a treatment modality.Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

[0094] Additionally, antibodies are useful in pharmacogenomic analysis.Thus, antibodies prepared against polymorphic proteins can be used toidentify individuals that require modified treatment modalities. Theantibodies are also useful as diagnostic tools as an immunologicalmarker for aberrant protein analyzed by electrophoretic mobility,isoelectric point, tryptic peptide digest, and other physical assaysknown to those in the art.

[0095] The antibodies are also useful for tissue typing. Experimentaldata as provided in FIG. 1 indicates expression in lung carcinoma andplacenta. Thus, where a specific protein has been correlated withexpression in a specific tissue, antibodies that are specific for thisprotein can be used to identify a tissue type.

[0096] The antibodies are also useful for inhibiting protein function,for example, blocking the binding of the kinase peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

[0097] The invention also encompasses kits for using antibodies todetect the presence of a protein in a biological sample. The kit cancomprise antibodies such as a labeled or labelable antibody and acompound or agent for detecting protein in a biological sample; meansfor determining the amount of protein in the sample; means for comparingthe amount of protein in the sample with a standard; and instructionsfor use. Such a kit can be supplied to detect a single protein orepitope or can be configured to detect one of a multitude of epitopes,such as in an antibody detection array. Arrays are described in detailbelow for nuleic acid arrays and similar methods have been developed forantibody arrays.

[0098] Nucleic Acid Molecules

[0099] The present invention further provides isolated nucleic acidmolecules that encode a kinase peptide or protein of the presentinvention (cDNA, transcript and genomic sequence). Such nucleic acidmolecules will consist of, consist essentially of, or comprise anucleotide sequence that encodes one of the kinase peptides of thepresent invention, an allelic variant thereof, or an ortholog or paralogthereof.

[0100] As used herein, an “isolated” nucleic acid molecule is one thatis separated from other nucleic acid present in the natural source ofthe nucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

[0101] Moreover, an “isolated” nucleic acid molecule, such as atranscript/cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orchemical precursors or other chemicals when chemically synthesized.However, the nucleic acid molecule can be fused to other coding orregulatory sequences and still be considered isolated.

[0102] For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

[0103] Accordingly, the present invention provides nucleic acidmolecules that consist of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists of a nucleotide sequencewhen the nucleotide sequence is the complete nucleotide sequence of thenucleic acid molecule.

[0104] The present invention further provides nucleic acid moleculesthat consist essentially of the nucleotide sequence shown in FIG. 1 or 3(SEQ ID NO:1, transcript sequence and SEQ ID NO:3, genomic sequence), orany nucleic acid molecule that encodes the protein provided in FIG. 2,SEQ ID NO:2. A nucleic acid molecule consists essentially of anucleotide sequence when such a nucleotide sequence is present with onlya few additional nucleic acid residues in the final nucleic acidmolecule.

[0105] The present invention further provides nucleic acid moleculesthat comprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ IDNO:1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule comprises a nucleotide sequence whenthe nucleotide sequence is at least part of the final nucleotidesequence of the nucleic acid molecule. In such a fashion, the nucleicacid molecule can be only the nucleotide sequence or have additionalnucleic acid residues, such as nucleic acid residues that are naturallyassociated with it or heterologous nucleotide sequences. Such a nucleicacid molecule can have a few additional nucleotides or can comprisesseveral hundred or more additional nucleotides. A brief description ofhow various types of these nucleic acid molecules can be readilymade/isolated is provided below.

[0106] In FIGS. 1 and 3, both coding and non-coding sequences areprovided. Because of the source of the present invention, humans genomicsequence (FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleicacid molecules in the Figures will contain genomic intronic sequences,5′ and 3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

[0107] The isolated nucleic acid molecules can encode the mature proteinplus additional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

[0108] As mentioned above, the isolated nucleic acid molecules include,but are not limited to, the sequence encoding the kinase peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

[0109] Isolated nucleic acid molecules can be in the form of RNA, suchas mRNA, or in the form DNA, including cDNA and genomic DNA obtained bycloning or produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

[0110] The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the kinase proteins ofthe present invention that are described above. Such nucleic acidmolecules may be naturally occurring, such as allelic variants (samelocus), paralogs (different locus), and orthologs (different organism),or may be constructed by recombinant DNA methods or by chemicalsynthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

[0111] The present invention further provides non-coding fragments ofthe nucleic acid molecules provided in FIGS. 1 and 3. Preferrednon-coding fragments include, but are not limited to, promotersequences, enhancer sequences, gene modulating sequences and genetermination sequences. Such fragments are useful in controllingheterologous gene expression and in developing screens to identifygene-modulating agents. A promoter can readily be identified as being 5′to the ATG start site in the genomic sequence provided in FIG. 3.

[0112] A fragment comprises a contiguous nucleotide sequence greaterthan 12 or more nucleotides. Further, a fragment could at least 30, 40,50, 100, 250 or 500 nucleotides in length. The length of the fragmentwill be based on its intended use. For example, the fragment can encodeepitope bearing regions of the peptide, or can be useful as DNA probesand primers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

[0113] A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

[0114] Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. As indicated by thedata presented in FIG. 3, the map position was determined to be onchromosome 13 by ePCR, and confirmed with radiation hybrid mapping.

[0115]FIG. 3 provides information on SNPs that have been found in a geneencoding the kinase proteins of the present invention. Thirty-three SNPswere identified. The changes in the amino acid sequence that these SNPscause can readily be determined using the universal genetic code and theprotein sequence provided in FIG. 2 as a base.

[0116] As used herein, the term “hybridizes under stringent conditions”is intended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

[0117] Nucleic Acid Molecule Uses

[0118] The nucleic acid molecules of the present invention are usefulfor probes, primers, chemical intermediates, and in biological assays.The nucleic acid molecules are useful as a hybridization probe formessenger RNA, transcript/cDNA and genomic DNA to isolate full-lengthcDNA and genomic clones encoding the peptide described in FIG. 2 and toisolate cDNA and genomic clones that correspond to variants (alleles,orthologs, etc.) producing the same or related peptides shown in FIG. 2.As illustrated in FIG. 3, SNPs were identified at 33 positions.

[0119] The probe can correspond to any sequence along the entire lengthof the nucleic acid molecules provided in the Figures. Accordingly, itcould be derived from 5′ noncoding regions, the coding region, and 3′noncoding regions. However, as discussed, fragments are not to beconstrued as encompassing fragments disclosed prior to the presentinvention.

[0120] The nucleic acid molecules are also useful as primers for PCR toamplify any given region of a nucleic acid molecule and are useful tosynthesize antisense molecules of desired length and sequence.

[0121] The nucleic acid molecules are also useful for constructingrecombinant vectors. Such vectors include expression vectors thatexpress a portion of, or all of, the peptide sequences. Vectors alsoinclude insertion vectors, used to integrate into another nucleic acidmolecule sequence, such as into the cellular genome, to alter in situexpression of a gene and/or gene product. For example, an endogenouscoding sequence can be replaced via homologous recombination with all orpart of the coding region containing one or more specifically introducedmutations.

[0122] The nucleic acid molecules are also useful for expressingantigenic portions of the proteins.

[0123] The nucleic acid molecules are also useful as probes fordetermining the chromosomal positions of the nucleic acid molecules bymeans of in situ hybridization methods. As indicated by the datapresented in FIG. 3, the map position was determined to be on chromosome13 by ePCR, and confirmed with radiation hybrid mapping.

[0124] The nucleic acid molecules are also useful in making vectorscontaining the gene regulatory regions of the nucleic acid molecules ofthe present invention.

[0125] The nucleic acid molecules are also useful for designingribozymes corresponding to all, or a part, of the mRNA produced from thenucleic acid molecules described herein.

[0126] The nucleic acid molecules are also useful for making vectorsthat express part, or all, of the peptides.

[0127] The nucleic acid molecules are also useful for constructing hostcells expressing a part, or all, of the nucleic acid molecules andpeptides.

[0128] The nucleic acid molecules are also useful for constructingtransgenic animals expressing all, or a part, of the nucleic acidmolecules and peptides.

[0129] The nucleic acid molecules are also useful as hybridizationprobes for determining the presence, level, form and distribution ofnucleic acid expression. Experimental data as provided in FIG. 1indicates that kinase proteins of the present invention are expressed inlung carcinoma, as indicated by virtual northern blot analysis, andplacenta, which is the cDNA library source from which the cDNA clonecame from. Accordingly, the probes can be used to detect the presenceof, or to determine levels of, a specific nucleic acid molecule incells, tissues, and in organisms. The nucleic acid whose level isdetermined can be DNA or RNA. Accordingly, probes corresponding to thepeptides described herein can be used to assess expression and/or genecopy number in a given cell, tissue, or organism. These uses arerelevant for diagnosis of disorders involving an increase or decrease inkinase protein expression relative to normal results.

[0130] In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

[0131] Probes can be used as a part of a diagnostic test kit foridentifying cells or tissues that express a kinase protein, such as bymeasuring a level of a kinase-encoding nucleic acid in a sample of cellsfrom a subject e.g., mRNA or genomic DNA, or determining if a kinasegene has been mutated. Experimental data as provided in FIG. 1 indicatesthat kinase proteins of the present invention are expressed in lungcarcinoma, as indicated by virtual northern blot analysis, and placenta,which is the cDNA library source from which the cDNA clone came from.

[0132] Nucleic acid expression assays are useful for drug screening toidentify compounds that modulate kinase nucleic acid expression.

[0133] The invention thus provides a method for identifying a compoundthat can be used to treat a disorder associated with nucleic acidexpression of the kinase gene, particularly biological and pathologicalprocesses that are mediated by the kinase in cells and tissues thatexpress it. Experimental data as provided in FIG. 1 indicates expressionin lung carcinoma and placenta. The method typically includes assayingthe ability of the compound to modulate the expression of the kinasenucleic acid and thus identifying a compound that can be used to treat adisorder characterized by undesired kinase nucleic acid expression. Theassays can be performed in cell-based and cell-free systems. Cell-basedassays include cells naturally expressing the kinase nucleic acid orrecombinant cells genetically engineered to express specific nucleicacid sequences.

[0134] The assay for kinase nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the kinase proteinsignal pathway can also be assayed. In this embodiment the regulatoryregions of these genes can be operably linked to a reporter gene such asluciferase.

[0135] Thus, modulators of kinase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of kinase mRNA inthe presence of the candidate compound is compared to the level ofexpression of kinase mRNA in the absence of the candidate compound. Thecandidate compound can then be identified as a modulator of nucleic acidexpression based on this comparison and be used, for example to treat adisorder characterized by aberrant nucleic acid expression. Whenexpression of mRNA is statistically significantly greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of nucleic acid expression. Whennucleic acid expression is statistically significantly less in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of nucleic acid expression.

[0136] The invention further provides methods of treatment, with thenucleic acid as a target, using a compound identified through drugscreening as a gene modulator to modulate kinase nucleic acid expressionin cells and tissues that express the kinase. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in lung carcinoma, as indicated by virtualnorthern blot analysis, and placenta, which is the cDNA library sourcefrom which the cDNA clone came from. Modulation includes bothup-regulation (i.e. activation or agonization) or down-regulation(suppression or antagonization) or nucleic acid expression.

[0137] Alternatively, a modulator for kinase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thekinase nucleic acid expression in the cells and tissues that express theprotein. Experimental data as provided in FIG. 1 indicates expression inlung carcinoma and placenta.

[0138] The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe kinase gene in clinical trials or in a treatment regimen. Thus, thegene expression pattern can serve as a barometer for the continuingeffectiveness of treatment with the compound, particularly withcompounds to which a patient can develop resistance. The gene expressionpattern can also serve as a marker indicative of a physiologicalresponse of the affected cells to the compound. Accordingly, suchmonitoring would allow either increased administration of the compoundor the administration of alternative compounds to which the patient hasnot become resistant. Similarly, if the level of nucleic acid expressionfalls below a desirable level, administration of the compound could becommensurately decreased.

[0139] The nucleic acid molecules are also useful in diagnostic assaysfor qualitative changes in kinase nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in kinase genes and geneexpression products such as mRNA. The nucleic acid molecules can be usedas hybridization probes to detect naturally occurring genetic mutationsin the kinase gene and thereby to determine whether a subject with themutation is at risk for a disorder caused by the mutation. Mutationsinclude deletion, addition, or substitution of one or more nucleotidesin the gene, chromosomal rearrangement, such as inversion ortransposition, modification of genomic DNA, such as aberrant methylationpatterns or changes in gene copy number, such as amplification.Detection of a mutated form of the kinase gene associated with adysfunction provides a diagnostic tool for an active disease orsusceptibility to disease when the disease results from overexpression,underexpression, or altered expression of a kinase protein.

[0140] Individuals carrying mutations in the kinase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in a gene encoding the kinaseproteins of the present invention. Thirty-three SNPs were identified.The changes in the amino acid sequence that these SNPs cause can readilybe determined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a base. As indicated by the data presented in FIG.3, the map position was determined to be on chromosome 13 by ePCR, andconfirmed with radiation hybrid mapping. Genomic DNA can be analyzeddirectly or can be amplified by using PCR prior to analysis. RNA or cDNAcan be used in the same way. In some uses, detection of the mutationinvolves the use of a probe/primer in a polymerase chain reaction (PCR)(see, e.g. U.S. Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCRor RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see,e.g., Landegran et al., Science 241:1077-1080 (1988); and Nakazawa etal., PNAS 91:360-364 (1994)), the latter of which can be particularlyuseful for detecting point mutations in the gene (see Abravaya et al.,Nucleic Acids Res. 23:675-682 (1995)). This method can include the stepsof collecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

[0141] Alternatively, mutations in a kinase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

[0142] Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531)can be used to score for the presence of specific mutations bydevelopment or loss of a ribozyme cleavage site. Perfectly matchedsequences can be distinguished from mismatched sequences by nucleasecleavage digestion assays or by differences in melting temperature.

[0143] Sequence changes at specific locations can also be assessed bynuclease protection assays such as RNase and S1 protection or thechemical cleavage method. Furthermore, sequence differences between amutant kinase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al., Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

[0144] Other methods for detecting mutations in the gene include methodsin which protection from cleavage agents is used to detect mismatchedbases in RNA/RNA or RNA/DNA duplexes (Myers et al., Science 230:1242(1985)); Cotton et al., PNAS 85:4397 (1988); Saleeba et al., Meth.Enzymol. 217:286-295 (1992)), electrophoretic mobility of mutant andwild type nucleic acid is compared (Orita et al., PNAS 86:2766 (1989);Cotton et al., Mutat. Res. 285:125-144 (1993); and Hayashi et al.,Genet. Anal. Tech. Appl. 9:73-79 (1992)), and movement of mutant orwild-type fragments in polyacrylamide gels containing a gradient ofdenaturant is assayed using denaturing gradient gel electrophoresis(Myers et al., Nature 313:495 (1985)). Examples of other techniques fordetecting point mutations include selective oligonucleotidehybridization, selective amplification, and selective primer extension.

[0145] The nucleic acid molecules are also useful for testing anindividual for a genotype that while not necessarily causing thedisease, nevertheless affects the treatment modality. Thus, the nucleicacid molecules can be used to study the relationship between anindividual's genotype and the individual's response to a compound usedfor treatment (pharmacogenomic relationship). Accordingly, the nucleicacid molecules described herein can be used to assess the mutationcontent of the kinase gene in an individual in order to select anappropriate compound or dosage regimen for treatment. FIG. 3 providesinformation on SNPs that have been found in a gene encoding the kinaseproteins of the present invention. Thirty-three SNPs were identified.The changes in the amino acid sequence that these SNPs cause can readilybe determined using the universal genetic code and the protein sequenceprovided in FIG. 2 as a base.

[0146] Thus nucleic acid molecules displaying genetic variations thataffect treatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

[0147] The nucleic acid molecules are thus useful as antisenseconstructs to control kinase gene expression in cells, tissues, andorganisms. A DNA antisense nucleic acid molecule is designed to becomplementary to a region of the gene involved in transcription,preventing transcription and hence production of kinase protein. Anantisense RNA or DNA nucleic acid molecule would hybridize to the mRNAand thus block translation of mRNA into kinase protein.

[0148] Alternatively, a class of antisense molecules can be used toinactivate mRNA in order to decrease expression of kinase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired kinase nucleic acid expression. This techniqueinvolves cleavage by means of ribozymes containing nucleotide sequencescomplementary to one or more regions in the mRNA that attenuate theability of the mRNA to be translated. Possible regions include codingregions and particularly coding regions corresponding to the catalyticand other functional activities of the kinase protein, such as substratebinding.

[0149] The nucleic acid molecules also provide vectors for gene therapyin patients containing cells that are aberrant in kinase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desired kinaseprotein to treat the individual.

[0150] The invention also encompasses kits for detecting the presence ofa kinase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that kinase proteins of the presentinvention are expressed in lung carcinoma, as indicated by virtualnorthern blot analysis, and placenta, which is the cDNA library sourcefrom which the cDNA clone came from. For example, the kit can comprisereagents such as a labeled or labelable nucleic acid or agent capable ofdetecting kinase nucleic acid in a biological sample; means fordetermining the amount of kinase nucleic acid in the sample; and meansfor comparing the amount of kinase nucleic acid in the sample with astandard. The compound or agent can be packaged in a suitable container.The kit can further comprise instructions for using the kit to detectkinase protein mRNA or DNA.

[0151] Nucleic Acid Arrays

[0152] The present invention further provides nucleic acid detectionkits, such as arrays or microarrays of nucleic acid molecules that arebased on the sequence information provided in FIGS. 1 and 3 (SEQ IDNOS:1 and 3).

[0153] As used herein “Arrays” or “Microarrays” refers to an array ofdistinct polynucleotides or oligonucleotides synthesized on a substrate,such as paper, nylon or other type of membrane, filter, chip, glassslide, or any other suitable solid support. In one embodiment, themicroarray is prepared and used according to the methods described inU.S. Pat. No. 5,837,832, Chee et al., PCT application WO95/11995 (Cheeet al.), Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) andSchena, M. et al. (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all ofwhich are incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

[0154] The microarray or detection kit is preferably composed of a largenumber of unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about6-60.nucleotides in-length, more preferably 15-30 nucleotides in length,and most preferably about. 20-25 nucleotides in length. For a certaintype of microarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

[0155] In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

[0156] In another aspect, an oligonucleotide may be synthesized on thesurface of the substrate by using a chemical coupling procedure and anink jet application apparatus, as described in PCT applicationWO95/251116 (Baldeschweiler et al.) which is incorporated herein in itsentirety by reference. In another aspect, a “gridded” array analogous toa dot (or slot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

[0157] In order to conduct sample analysis using a microarray ordetection kit, the RNA or DNA from a biological sample is made intohybridization probes. The mRNA is isolated, and cDNA is produced andused as a template to make antisense RNA (aRNA). The aRNA is amplifiedin the presence of fluorescent nucleotides, and labeled probes areincubated with the microarray or detection kit so that the probesequences hybridize to complementary oligonucleotides of the microarrayor detection kit. Incubation conditions are adjusted so thathybridization occurs with precise complementary matches or with variousdegrees of less complementarity. After removal of nonhybridized probes,a scanner is used to determine the levels and patterns of fluorescence.The scanned images are examined to determine degree of complementarityand the relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

[0158] Using such arrays, the present invention provides methods toidentify the expression of the kinase proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of the kinasegene of the present invention. FIG. 3 provides information on SNPs thathave been found in a gene encoding the kinase proteins of the presentinvention. Thirty-three SNPs were identified. The changes in the aminoacid sequence that these SNPs cause can readily be determined using theuniversal genetic code and the protein sequence provided in FIG. 2 as abase.

[0159] Conditions for incubating a nucleic acid molecule with a testsample vary. Incubation conditions depend on the format employed in theassay, the detection methods employed, and the type and nature of thenucleic acid molecule used in the assay. One skilled in the art willrecognize that any one of the commonly available hybridization,amplification or array assay formats can readily be adapted to employthe novel fragments of the Human genome disclosed herein. Examples ofsuch assays can be found in Chard, T, An Introduction toRadioimmunoassay and Related Techniques, Elsevier Science Publishers,Amsterdam, The Netherlands (1986); Bullock, G. R. et al., Techniques inImmunocytochemistry, Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2(1983), Vol. 3 (1985); Tijssen, P., Practice and Theory of EnzymeImmunoassays: Laboratory Techniques in Biochemistry and MolecularBiology, Elsevier Science Publishers, Amsterdam, The Netherlands (1985).

[0160] The test samples of the present invention include cells, proteinor membrane extracts of cells. The test sample used in theabove-described method will vary based on the assay format, nature ofthe detection method and the tissues, cells or extracts used as thesample to be assayed. Methods for preparing nucleic acid extracts or ofcells are well known in the art and can be readily be adapted in orderto obtain a sample that is compatible with the system utilized.

[0161] In another embodiment of the present invention, kits are providedwhich contain the necessary reagents to carry out the assays of thepresent invention.

[0162] Specifically, the invention provides a compartmentalized kit toreceive, in close confinement, one or more containers which comprises:(a) a first container comprising one of the nucleic acid molecules thatcan bind to a fragment of the Human genome disclosed herein; and (b) oneor more other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

[0163] In detail, a compartmentalized kit includes any kit in whichreagents are contained in separate containers. Such containers includesmall glass containers, plastic containers, strips of plastic, glass orpaper, or arraying material such as silica. Such containers allows oneto efficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified kinase gene of the present invention can beroutinely identified using the sequence information disclosed herein canbe readily incorporated into one of the established kit formats whichare well known in the art, particularly expression arrays.

[0164] Vectors/Host Cells

[0165] The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

[0166] A vector can be maintained in the host cell as anextrachromosomal element where it replicates and produces additionalcopies of the nucleic acid molecules. Alternatively, the vector mayintegrate into the host cell genome and produce additional copies of thenucleic acid molecules when the host cell replicates.

[0167] The invention provides vectors for the maintenance (cloningvectors) or vectors for expression (expression vectors) of the nucleicacid molecules. The vectors can function in prokaryotic or eukaryoticcells or in both (shuttle vectors).

[0168] Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

[0169] The regulatory sequence to which the nucleic acid moleculesdescribed herein can be operably linked include promoters for directingmRNA transcription. These include, but are not limited to, the leftpromoter from bacteriophage λ, the lac, TRP, and TAC promoters from E.Coli, the early and late promoters from SV40, the CMV immediate earlypromoter, the adenovirus early and late promoters, and retroviruslong-terminal repeats.

[0170] In addition to control regions that promote transcription,expression vectors may also include regions that modulate transcription,such as repressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

[0171] In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual.2nd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

[0172] A variety of expression vectors can be used to express a nucleicacid molecule. Such vectors include chromosomal, episomal, andvirus-derived vectors, for example vectors derived from bacterialplasmids, from bacteriophage, from yeast episomes, from yeastchromosomal elements, including yeast artificial chromosomes, fromviruses such as baculoviruses, papovaviruses such as SV40, Vacciniaviruses, adenoviruses, poxviruses, pseudorabies viruses, andretroviruses. Vectors may also be derived from combinations of thesesources such as those derived from plasmid and bacteriophage geneticelements, e.g. cosmids and phagemids. Appropriate cloning and expressionvectors for prokaryotic and eukaryotic hosts are described in Sambrooket al., Molecular Cloning: A Laboratory Manual. 2nd. ed., Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., (1989).

[0173] The regulatory sequence may provide constitutive expression inone or more host cells (i.e. tissue specific) or may provide forinducible expression in one or more cell types such as by temperature,nutrient additive, or exogenous factor such as a hormone or otherligand. A variety of vectors providing for constitutive and inducibleexpression in prokaryotic and eukaryotic hosts are well known to thoseof ordinary skill in the art.

[0174] The nucleic acid molecules can be inserted into the vectornucleic acid by well-known methodology. Generally, the DNA sequence thatwill ultimately be expressed is joined to an expression vector bycleaving the DNA sequence and the expression vector with one or morerestriction enzymes and then ligating the fragments together. Proceduresfor restriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

[0175] The vector containing the appropriate nucleic acid molecule canbe introduced into an appropriate host cell for propagation orexpression using well-known techniques. Bacterial cells include, but arenot limited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

[0176] As described herein, it may be desirable to express the peptideas a fusion protein. Accordingly, the invention provides fusion vectorsthat allow for the production of the peptides. Fusion vectors canincrease the expression of a recombinant protein, increase thesolubility of the recombinant protein, and aid in the purification ofthe protein by acting for example as a ligand for affinity purification.A proteolytic cleavage site may be introduced at the junction of thefusion moiety so that the desired peptide can ultimately be separatedfrom the fusion moiety. Proteolytic enzymes include, but are not limitedto, factor Xa, thrombin, and enterokinase. Typical fusion expressionvectors include pGEX (Smith et al., Gene 67:3140 (1988)), pMAL (NewEngland Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.)which fuse glutathione S-transferase (GST), maltose E binding protein,or protein A, respectively, to the target recombinant protein. Examplesof suitable inducible non-fusion E. coli expression vectors include pTrc(Amann et al., Gene 69:301-315 (1988)) and pET 11d (Studier et al., GeneExpression Technology: Methods in Enzymology 185:60-89 (1990)).

[0177] Recombinant protein expression can be maximized in host bacteriaby providing a genetic background wherein the host cell has an impairedcapacity to proteolytically cleave the recombinant protein. (Gottesman,S., Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990) 119-128). Alternatively, the sequence ofthe nucleic acid molecule of interest can be altered to providepreferential codon usage for a specific host cell, for example E. coli.(Wada et al., Nucleic Acids Res. 20:2111-2118 (1992)).

[0178] The nucleic acid molecules can also be expressed by expressionvectors that are operative in yeast. Examples of vectors for expressionin yeast e.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J.6:229-234 (1987)), pMFa (Kurjan et al., Cell 30:933-943(1982)), pJRY88(Schultz et al., Gene 54:113-123 (1987)), and pYES2 (InvitrogenCorporation, San Diego, Calif.).

[0179] The nucleic acid molecules can also be expressed in insect cellsusing, for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

[0180] In certain embodiments of the invention, the nucleic acidmolecules described herein are expressed in mammalian cells usingmammalian expression vectors. Examples of mammalian expression vectorsinclude pCDM8 (Seed, B. Nature 329:840(1987)) and pMF2PC (Kaufman etal., EMBO J. 6:187-195 (1987)).

[0181] The expression vectors listed herein are provided by way ofexample only of the well-known vectors available to those of ordinaryskill in the art that would be useful to express the nucleic acidmolecules. The person of ordinary skill in the art would be aware ofother vectors suitable for maintenance propagation or expression of thenucleic acid molecules described herein. These are found for example inSambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: ALaboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

[0182] The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

[0183] The invention also relates to recombinant host cells containingthe vectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

[0184] The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

[0185] Host cells can contain more than one vector. Thus, differentnucleotide sequences can be introduced on different vectors of the samecell. Similarly, the nucleic acid molecules can be introduced eitheralone or with other nucleic acid molecules that are not related to thenucleic acid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

[0186] In the case of bacteriophage and viral vectors, these can beintroduced into cells as packaged or encapsulated virus by standardprocedures for infection and transduction. Viral vectors can bereplication-competent or replication-defective. In the case in whichviral replication is defective, replication will occur in host cellsproviding functions that complement the defects.

[0187] Vectors generally include selectable markers that enable theselection of the subpopulation of cells that contain the recombinantvector constructs. The marker can be contained in the same vector thatcontains the nucleic acid molecules described herein or may be on aseparate vector. Markers include tetracycline or ampicillin-resistancegenes for prokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

[0188] While the mature proteins can be produced in bacteria, yeast,mammalian cells, and other cells under the control of the appropriateregulatory sequences, cell-free transcription and translation systemscan also be used to produce these proteins using RNA derived from theDNA constructs described herein.

[0189] Where secretion of the peptide is desired, which is difficult toachieve with multi-transmembrane domain containing proteins such askinases, appropriate secretion signals are incorporated into the vector;The signal sequence can be endogenous to the peptides or heterologous tothese peptides.

[0190] Where the peptide is not secreted into the medium, which istypically the case with kinases, the protein can be isolated from thehost cell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

[0191] It is also understood that depending upon the host cell inrecombinant production of the peptides described herein, the peptidescan have various glycosylation patterns, depending upon the cell, ormaybe non-glycosylated as when produced in bacteria. In addition, thepeptides may include an initial modified methionine in some cases as aresult of a host-mediated process.

[0192] Uses of Vectors and Host Cells

[0193] The recombinant host cells expressing the peptides describedherein have a variety of uses. First, the cells are useful for producinga kinase protein or peptide that can be further purified to producedesired amounts of kinase protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production

[0194] Host cells are also useful for conducting cell-based assaysinvolving the kinase protein or kinase protein fragments, such as thosedescribed above as well as other formats known in the art. Thus, arecombinant host cell expressing a native kinase protein is useful forassaying compounds that stimulate or inhibit kinase protein function.

[0195] Host cells are also useful for identifying kinase protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantkinase protein (for example, stimulating or inhibiting function) whichmay not be indicated by their effect on the native kinase protein.

[0196] Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a kinase proteinand identifying and evaluating modulators of kinase protein activity.Other examples of transgenic animals include non-human primates, sheep,dogs, cows, goats, chickens, and amphibians.

[0197] A transgenic animal can be produced by introducing nucleic acidinto the male pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the kinase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

[0198] Any of the regulatory or other sequences useful in expressionvectors can form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the kinase protein to particularcells.

[0199] Methods for generating transgenic animals via embryo manipulationand microinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al., U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

[0200] In another embodiment, transgenic non-human animals can beproduced which contain selected systems that allow for regulatedexpression of the transgene. One example of such a system is thecre/loxP recombinase system of bacteriophage P1. For a description ofthe cre/loxP recombinase system, see, e.g., Lakso et al. PNAS89:6232-6236 (1992). Another example of a recombinase system is the FLPrecombinase system of S. cerevisiae (O'Gorman et al. Science251:1351-1355 (1991). If a cre/loxP recombinase system is used toregulate expression of the transgene, animals containing transgenesencoding both the Cre recombinase and a selected protein is required.Such animals can be provided through the construction of “double”transgenic animals, e.g., by mating two transgenic animals, onecontaining a transgene encoding a selected protein and the othercontaining a transgene encoding a recombinase.

[0201] Clones of the non-human transgenic animals described herein canalso be produced according to the methods described in Wilmut, I. et al.Nature 385:810-813 (1997) and PCT International Publication Nos. WO97/07668 and WO 97/07669. In brief, a cell, e.g., a somatic cell, fromthe transgenic animal can be isolated and induced to exit the growthcycle and enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

[0202] Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, kinase protein activation, and signal transduction, may not beevident from in vitro cell-free or cell-based assays. Accordingly, it isuseful to provide non-human transgenic animals to assay in vivo kinaseprotein function, including substrate interaction, the effect ofspecific mutant kinase proteins on kinase protein function and substrateinteraction, and the effect of chimeric kinase proteins. It is alsopossible to assess the effect of null mutations, that is, mutations thatsubstantially or completely eliminate one or more kinase proteinfunctions.

[0203] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of theabove-described modes for carrying out the invention which are obviousto those skilled in the field of molecular biology or related fields areintended to be within the scope of the following claims.

1 4 1 2370 DNA Homo sapiens 1 cattggagac catggataag tacgatgtgattaaggccat cgggcaaggt gccttcggga 60 aagcatactt agctaaaggg aaatcagatagcaagcactg tgtcataaaa gagatcaatt 120 ttgaaaagat gcccatacaa gaaaaagaagcttcaaagaa agaagtgatt cttctggaaa 180 agatgaaaca tcccaacatt gtagccttcttcaattcatt tcaagagaat ggcaggctgt 240 ttattgtaat ggaatattgt gatggaggggatctcatgaa aaggatcaat agacaacggg 300 gtgtgttatt tagtgaagat cagatcctcggttggtttgt acagatttct ctaggactaa 360 aacatattca tgacaggaag atattacacagggacataaa agctcagaac atttttctta 420 gcaagaacgg aatggtggca aagcttggggactttggtat agcaagagtc ctgaataatt 480 ccatggaact tgctcgaact tgtattggaacaccttacta cctgtcccca gagatctgtc 540 agaataaacc ctacaacaat aaaacggatatttggtctct tggctgtgtc ttatatgagc 600 tctgcacact taaacatcct tttgagggtaacaacttaca gcagctggtt ctgaagattt 660 gtcaagcaca ttttgcccca atatctccggggttttctcg tgagctccat tccttgatat 720 ctcagctctt tcaagtatct cctcgagaccgaccatccat aaattccatt ttgaaaaggc 780 cctttttaga gaatcttatt cccaaatatttgactcctga ggtcattcag gaagaattca 840 gtcacatgct tatatgcaga gcaggagcgccagcttctcg acatgctggg aaggtggtcc 900 agaagtgtaa aatacaaaaa gtgagattccggggaaagtg cccaccaaga tcaaggatat 960 ctgtgccaat taaaaggaat gctatattgcatagaaatga atggagacca ccagctggag 1020 cccagaaggc cagatctata aaaatgatagaaagacccaa aattgctgct gtctgtggac 1080 attatgatta ttattatgct caacttgatatgctgaggag gagagcccac aaaccaagtt 1140 atcaccctat tcctcaagaa aatactggagttgaggatta cggtcaggaa acgaggcatg 1200 gtccatcccc aagtcaatgg cctgctgagtaccttcagag aaaatttgaa gctcaacaat 1260 ataagttgaa agtggagaag caattgggtcttcgtccatc ttctgccgag ccaaattaca 1320 accagagaca agagctaaga agtaatggagaagagcctag attccaggag ctgccattta 1380 ggaaaaacga aatgaaggaa caggaatattggaagcagtt agaggaaata cgccaacagt 1440 acctcaatga catgaaagaa attagaaagaagatggggag agaaccagag gacattgaaa 1500 aagacttgaa acaaatgagg cttcagaacacaaaggaaag taaaaatcca gaacagaaat 1560 ataaagctaa gaagggggta aaatttgaaattaatttaga caaatgtatt tctgatgaaa 1620 acatcctcca agaggaagag gcaatggatataccaaatga aactttgacc tttgaggatg 1680 gcatgaagtt taaggaatat gaatgtgtaaaggagcatgg agattataca gacaaagcat 1740 ttgaaaaact tcactgccca gaagcagcatttacagaact gacttggctc agtttcctct 1800 tcctggaata ctctctgcct catttccttctggaaaaatc tccattcagc aggcatctta 1860 ttgaggatct cctttgtgcc aacgactgctcactgaagga ctggagtgag aaggaaatgg 1920 agcttaggac ataaccctac cactacataaacaaactttg gagaatcagg agagagtaaa 1980 gccaaaggag gagagacagg tcatggggaggcacaggaat tggcagcatc aactggaaga 2040 gaaaggccag atgagggttt tccacgcagactgtagttgc tgtgggaaac aggaggcagt 2100 gggatggagg agcgcctcag actctgctgcagatgatggc agtggccgac atcacctcca 2160 cctgccccac ggggcctgac agtgagtctgtgcttagtgt cagtcgtcag gaagggaaga 2220 ccaaggaccc gtacagccca gtgctcatcctgatgtgata gtctacttct cactatacac 2280 cctatagatc ttgtatcaga cactttcaaatatgttgttt tgatatctca agaaaaaaaa 2340 aaaaaaaaca ctgtcatgcc gttacgagcg2370 2 640 PRT Homo sapiens 2 Met Asp Lys Tyr Asp Val Ile Lys Ala IleGly Gln Gly Ala Phe Gly 1 5 10 15 Lys Ala Tyr Leu Ala Lys Gly Lys SerAsp Ser Lys His Cys Val Ile 20 25 30 Lys Glu Ile Asn Phe Glu Lys Met ProIle Gln Glu Lys Glu Ala Ser 35 40 45 Lys Lys Glu Val Ile Leu Leu Glu LysMet Lys His Pro Asn Ile Val 50 55 60 Ala Phe Phe Asn Ser Phe Gln Glu AsnGly Arg Leu Phe Ile Val Met 65 70 75 80 Glu Tyr Cys Asp Gly Gly Asp LeuMet Lys Arg Ile Asn Arg Gln Arg 85 90 95 Gly Val Leu Phe Ser Glu Asp GlnIle Leu Gly Trp Phe Val Gln Ile 100 105 110 Ser Leu Gly Leu Lys His IleHis Asp Arg Lys Ile Leu His Arg Asp 115 120 125 Ile Lys Ala Gln Asn IlePhe Leu Ser Lys Asn Gly Met Val Ala Lys 130 135 140 Leu Gly Asp Phe GlyIle Ala Arg Val Leu Asn Asn Ser Met Glu Leu 145 150 155 160 Ala Arg ThrCys Ile Gly Thr Pro Tyr Tyr Leu Ser Pro Glu Ile Cys 165 170 175 Gln AsnLys Pro Tyr Asn Asn Lys Thr Asp Ile Trp Ser Leu Gly Cys 180 185 190 ValLeu Tyr Glu Leu Cys Thr Leu Lys His Pro Phe Glu Gly Asn Asn 195 200 205Leu Gln Gln Leu Val Leu Lys Ile Cys Gln Ala His Phe Ala Pro Ile 210 215220 Ser Pro Gly Phe Ser Arg Glu Leu His Ser Leu Ile Ser Gln Leu Phe 225230 235 240 Gln Val Ser Pro Arg Asp Arg Pro Ser Ile Asn Ser Ile Leu LysArg 245 250 255 Pro Phe Leu Glu Asn Leu Ile Pro Lys Tyr Leu Thr Pro GluVal Ile 260 265 270 Gln Glu Glu Phe Ser His Met Leu Ile Cys Arg Ala GlyAla Pro Ala 275 280 285 Ser Arg His Ala Gly Lys Val Val Gln Lys Cys LysIle Gln Lys Val 290 295 300 Arg Phe Arg Gly Lys Cys Pro Pro Arg Ser ArgIle Ser Val Pro Ile 305 310 315 320 Lys Arg Asn Ala Ile Leu His Arg AsnGlu Trp Arg Pro Pro Ala Gly 325 330 335 Ala Gln Lys Ala Arg Ser Ile LysMet Ile Glu Arg Pro Lys Ile Ala 340 345 350 Ala Val Cys Gly His Tyr AspTyr Tyr Tyr Ala Gln Leu Asp Met Leu 355 360 365 Arg Arg Arg Ala His LysPro Ser Tyr His Pro Ile Pro Gln Glu Asn 370 375 380 Thr Gly Val Glu AspTyr Gly Gln Glu Thr Arg His Gly Pro Ser Pro 385 390 395 400 Ser Gln TrpPro Ala Glu Tyr Leu Gln Arg Lys Phe Glu Ala Gln Gln 405 410 415 Tyr LysLeu Lys Val Glu Lys Gln Leu Gly Leu Arg Pro Ser Ser Ala 420 425 430 GluPro Asn Tyr Asn Gln Arg Gln Glu Leu Arg Ser Asn Gly Glu Glu 435 440 445Pro Arg Phe Gln Glu Leu Pro Phe Arg Lys Asn Glu Met Lys Glu Gln 450 455460 Glu Tyr Trp Lys Gln Leu Glu Glu Ile Arg Gln Gln Tyr Leu Asn Asp 465470 475 480 Met Lys Glu Ile Arg Lys Lys Met Gly Arg Glu Pro Glu Asp IleGlu 485 490 495 Lys Asp Leu Lys Gln Met Arg Leu Gln Asn Thr Lys Glu SerLys Asn 500 505 510 Pro Glu Gln Lys Tyr Lys Ala Lys Lys Gly Val Lys PheGlu Ile Asn 515 520 525 Leu Asp Lys Cys Ile Ser Asp Glu Asn Ile Leu GlnGlu Glu Glu Ala 530 535 540 Met Asp Ile Pro Asn Glu Thr Leu Thr Phe GluAsp Gly Met Lys Phe 545 550 555 560 Lys Glu Tyr Glu Cys Val Lys Glu HisGly Asp Tyr Thr Asp Lys Ala 565 570 575 Phe Glu Lys Leu His Cys Pro GluAla Ala Phe Thr Glu Leu Thr Trp 580 585 590 Leu Ser Phe Leu Phe Leu GluTyr Ser Leu Pro His Phe Leu Leu Glu 595 600 605 Lys Ser Pro Phe Ser ArgHis Leu Ile Glu Asp Leu Leu Cys Ala Asn 610 615 620 Asp Cys Ser Leu LysAsp Trp Ser Glu Lys Glu Met Glu Leu Arg Thr 625 630 635 640 3 63588 DNAHomo sapiens misc_feature (1)...(63588) n = A,T,C or G 3 cttggcaggccgccgctgtg gcccaaagag taggaagccg ttccagtctc acgtccacct 60 tttggcaatatttgagacct tgtacaagaa acactcttcc tgtatcagtt tagctcattt 120 gtaaaactgggagactactg ccttgacggg ttgtaaagaa aagagagaac gtttgcgaag 180 cgtctggtgcaccttaagca agagcgggga gcgctactgt agactgcaaa gcaaaggaat 240 cccgacccaaggcaacggga cggttgcggg gtgactctgc cgggtctcca aactccctgg 300 cgcctgaccctgcctcgagg tggactggtc cccaggccat tccagacccg cgccccgccc 360 gcgtttccttgcgcggctcc gccccggccg cagggaggcg cagcggcccc gggaacccgg 420 atccttccgggacgcttcgt tggccccgcg gagccggcgg agcaggtacg cttgcagggg 480 ccgcccttagttcttgcccg gagccgccac agggcttcgg gagctcggca gggtggggga 540 aagggatggagtttcggcct ggggcggcgg gggcggccca gaaaaggcct agcgtcctgg 600 gctgtgtgggtgtagcgtcc agggcgcgtc ggtctctatg gcaacgctcc acacgcggag 660 gtcgggtacgggtaagcgtc ttgccactca cccgcggccg cttccagggg cggccctagg 720 ggagaaggaattttcctaat ttgggggctt ccaccctttg gtgccacttg ggcgggaggg 780 tcgcgggccctcagttcccg gcgagtcacc cccggcccca agtccgtatg cgtctctctc 840 agaacccgatcctccggtgt ctgcagcctc tcctggctgc ggagctggtt cccagccccc 900 tgcaacccagtaccgacttc ccaccctgac gtaaaattat tcgaaaacaa gccccctgct 960 caccccattaacaacaacaa caaaactgta ttatgcccta actgtagcat aaagaggaaa 1020 tagaaggaaagcaataagta agaaagtaca tatttcaatc tgaaaatgct tggcactact 1080 acccttggaaaatgtagaga agtagccagt agccgcgcct ggggagtcgc ctgaacgtga 1140 cggcagcaaatgcagattgt tgggtctccg ggaccaggag cagcgtggcc agtgaagcgc 1200 gtggttttcccaaatggtga acaattcttg gtaaacctcc aaaccgaagt gcaatcaagc 1260 cttgatttacatgtagttgc attcctcgaa aaaaaaaaga agtgttcatt aaaactgcaa 1320 aaatacttagcatttcgatg taaaatagag tttggttcta gccacaaacg gatttttcca 1380 cgcacaggaatgtataggaa gactctccaa gattgtaggg ccgcggggta atcctttatt 1440 gtgcgggactgtctctcgaa tcgcagaatc ctaccatctc aggccccaac cacctgtaaa 1500 cctcatgcctctgaatcttg gggaaacagc ttccccaccc ccatatattt ccagaattcc 1560 ccctagggggcagtacgtcc ccactaagaa aggctgaact ataaaagtgc acaagcctaa 1620 ggacattcctgctttataaa ggtgcgaaac accggatata gtatctttca ttctcagaac 1680 aaacttgcaaaacaggtatt gttattccat tttagaaatt aggaaagtga ggttttgcca 1740 ggttaagtgacttacccgag aatacagggc aaaagtgtat caaagctgag ctatgacccg 1800 tgtctgaccaagaaactctg tctcatttca gttatctgtg gccacaaaga aagttatttg 1860 tctctgtcttggcaaggctg ggaggaaagt tttagctaag tgagttcttt tacactttag 1920 tcatcagttttctgactttg ttagtcttta tgagacgtgt gtgataaatt tacattactc 1980 taattccaggaaactcagcc cattggagac catggataag tacgatgtga ttaaggccat 2040 cgggcaaggtgccttcggga aagcatactt agctaaaggg aaatcagata gcaagcactg 2100 tgtcataaaagagatcaatt ttgaaaaggt aaagttaagt tcaaatttct gttaattttc 2160 agtgggatattcagctggct tttaatccaa tataaaaagg aaatttttat tttttataat 2220 ttcgaattttaagccataat tgatttttgt taattcaacc tcctaagtcc attgtccaaa 2280 cagcaaccaatgatctcatt tttaaaaaga ggctggacgc actggctcac ccctgtaatc 2340 ccagcactttggatggccaa ggtgggagga ttgtgtgaag ccaggagttc gagatagcct 2400 gggcaacatagcaagaccct gtccctgcta aaaaaaaatt tttttaatga aaatagaaaa 2460 gaaataagatcacatccctg tggctcctat ggccctcctt agggtgccct gcaaggccct 2520 gtgagatgccagcctcctct gttgccctga cttttctctg tggtgcactt cctctctcct 2580 tattcaggtcctctacgagg ggttttctgc aaacatccta gctagagtag acccccagcc 2640 acaatcacaccttatcacct tatcacacca ccttggttcc tggtttcttt tttgttttct 2700 tttcttttctttttttagac ggagtctcgc tctgtcacct aggctggagt gcagtggcat 2760 gatcttggctcactgcaatc tccacctccg ggattcaagc aattctccca cttcagcctc 2820 ctgaatagctgggactacag gtgcatgcca ccatgcctgg ataatttttt gtatttttag 2880 tagagatggagtttcaccat gttgcccagg ctggtcttga actcctgagc tcaagtgatc 2940 tgcccgccttggcctcccaa agtgctggga ttacaggctt gagccactgc ttctggcctg 3000 gtttattttcttactagcat gtataatgct ctgcaattac tttgctctct taattattca 3060 tttgtttattgcttgtcttc ctcagtatgc agaacagttc ctgtcacata ataggtgcta 3120 aacacatttattgagtgcac tgaatgaata gagaaaaact atatgtaatt gttggtctaa 3180 tgattttggaaaataaatat agttaattaa aaattaataa tttttgctaa atccaccttg 3240 gtcagtgtttatgtcaccct ctttagtgat atgttcattt cataatatat tgggacaaca 3300 atgtccattgtttgctagaa ttaattctaa ggcaagtctt gttggtcagc ttctagagga 3360 tttataaatgagagtagcat aaaaagttcc atacaaagtg tgtgcaaaat ggactaccca 3420 agttacaccatatgaatata cttaatgcca ttgaactgta cacttaaaaa tcgttaaaat 3480 gatataaattttatcttacc acaaaaaatt gcaagaaaac ctacccaaac ttaaagctca 3540 agagtagatgactggcttcc agggataatg atttatttcc caatataggt ctctttttgt 3600 gaatccatggcatattcata ataatgtcct cttattctag tggcccgcaa tagcttcctc 3660 ccatgacattattctgctca ctctcttttg tttatctgac tgctctccct caggcttatc 3720 tctgtcttcgccctgtgtat gtcctcaacc atgtgtcctt ttctgatttt ctttttctgt 3780 ccattgtcacctaaactgcc ccacttcagt gtttaccaat aagtagatct ctcttaaatc 3840 tctgtctctacccctggcat ctttcagtac cctagttctg catttcttct gccagctaga 3900 taacttcaggtaatatctgt ggttttgttt tgaggtggag tctcgctctg tcgcccaggc 3960 tggagtgcagtggtgccatc tcggctcact gcaagctctg cctcccaggt tcatgccatt 4020 ctcctgcctcagcctcccga gtagctggga ctacaggcgc ccgccaccac gcctggctaa 4080 ttttttgtatttttagtaga gacggggttt cactgtgtta gctaggatgg tctcaatctc 4140 ctgacttcgtgatccacccg cctcggcctc ccaaagtgct gggattacag gcatgagcca 4200 ccacacccatccaacatcta tgttattaat ctattgctgt gtagcatatt accccaaact 4260 tagtggcttaaagaataaac atttattgtc tcagagatcc tgtgcatcaa gaatttagga 4320 tgacgatcattgagaccacc ttggaggctc ggtatcacaa ttgtacccaa aaacaagtat 4380 taatagtgattcttccttgt tgtaagcaga cccacttcac ctcctatgtg ctgcgctgta 4440 ttaatgtcatcagtgtcctt atggttgcca gcctgaaaac cttgggatcg tttgtgagct 4500 tattccttctccacattcaa ttatttggcg aatactgttg actcttcctc ttccttgaat 4560 ttgcttcagtccttttgtcg aggccctggg tcacttggat ccttcaagtg gctccagccc 4620 aattttgataatgctccagc catgccccca aaccttcact gggacagagg ctgtaaagaa 4680 agagttgcctaggtttgact acataaaaat agaaaacgtt tgtatgtcaa aacaaacact 4740 ataaataaattcaaagaaat cgagaaggtg ccaaaaatat ttgcaagtat tgacttaatg 4800 gtgttagccttttattaaat caataaaaag ataaaatcca tatatgaagt catcgtacaa 4860 aaatttgaaactcagtagaa aactaagaaa ttaggagttt attcaaagaa aaaccccaca 4920 gataaacagttagaaaacaa atgtccaaca gtaggtaatt tgttaagtaa tttataaaaa 4980 actaagtggctattagcaat catgttgtag gtgaagcatt gacatgggaa aatttcaatg 5040 tttgcaatgtttgagaaaat agtaagtgta aaataatata atctttggaa aaatatatat 5100 attctccatatatatgtata cctacaaata tgttcatata tgtacaaaga aagacacaaa 5160 ttgttattattgaggtagaa agtggggttt gccttgtgca ttttttttga gacaggattt 5220 cattctgttgcccaggcagg agtgcagtga catcatcatg gctcactgta accttgaaca 5280 agccatcctcctgcctcagc ctcctgagta gctaggactg caggcatgcg ccaccacacc 5340 cagctaattttgaaattgtt tttagagaca ggatcttgct atgttgccca ggctggcctc 5400 aagtgatcctcccactttgg cctcccaaag tgctgggatt acaggtgtta gccactgtgc 5460 ctggtctgccttgtgctttt atattgtttc actcttcaga gaagttttga gaccctctct 5520 gatttgctccaaaactacag ctcctatcac ataccctact ttttttcccc actccagcct 5580 ctgcatttgcttctggggct acttcttcca aggtcgttgc ctgctgatct cccagcatca 5640 agatcccacttgttcaaggc tgagctctac catacctcca gaatcctccc actctaaaga 5700 atttatccttctctgtaaac ttgcataact tttattggaa cctctgttat agtactgact 5760 gctttctttctggacatgct ttggctgttt attttgtgcc ttctcctcct tatttagctg 5820 taatatgttctgtgaggacc gagtccatgt gtgttttgtg ctggtattcc acacagcacc 5880 taatgcttggtgccaggaga tattcaataa cttcttattg gatagatgat tcactggaca 5940 gatgctttcaggccctcttg ctctactgtg aagctggtat atacttagga attataaaac 6000 cattttaattctatgtaaag agaaaatatt tgagaggtga atctctataa aaatgtacat 6060 taacattactgcatttcata gcatctctcc cattctttag tataatcaaa aattgactat 6120 atttttctaatagagcacca atttttcatc actttactca tgaactactc ttgtcactat 6180 gccataaataagtagaatct tatattagac ctcattattc ttgttttccc atatctgttt 6240 atgttatcgaatttacctat aacatctgtg tcacaatatt aacatttatt acttctttct 6300 tcctatctactctcatgtag tttttcatta cttcttatct agagaaattt atatttcttc 6360 tctctaatgcctccctactc cctacactag accccagaac taaattgctt gttttcttac 6420 aggtaccaaaaagctaatat ttctcttatc atcctaccat tatcaagcat gttcttttcc 6480 ttctgggctcaaataaaagt gttttatctt tcctcaattg tgaaaataaa aatgttcgtt 6540 gtagaaattttgaaaagagc caaaggagaa aataagacca tttagaggaa aataaaaata 6600 gcatataacctctttcttaa tcactatgaa cactttgctg aatttctctc tagactattt 6660 ttaatgtataagtatataag ttattagaat gattggtgtc atggtagata tactcttttt 6720 tttttttgagacggagtctc gctctgttgc ccaggctgga gtgcagtggc gtgatctctg 6780 ctcactgcaatctctgcctc ccaggttcaa acaattctcc tgcctcagcc tcctgagtag 6840 ctgggactacaggcgcatgc caccatgcct ggctaatttt tgtattttta gtagaaacag 6900 gctttcactatgttggccag gctggtctcg aactactgac ttcgtgatct gcctgccttg 6960 gcctcccaaagtgctgggat tacaggtgtg agccactgcg cccagcccag gtatactctt 7020 ttgtaacagttttttatatt agcaatatat tgtgaatatt tcctcatctc attcaatatt 7080 tttatataataaaatgttgt catttaatga tattaaatgt gttcacacta atgataaagg 7140 gaccacctgcagggtgtcca ttatatgtca caccatcctg ggtgttttat tatgtatatc 7200 aactcaatttaatcttcaca accacttaaa aggtagctct cattactctc actgtacaag 7260 tgaaagagctgaggctaaag aggttaagca gttagctcca ggatgcacag taatcagcag 7320 atccatctaagtctttctct gctctttcca tgatactaca ttgcctccct ttatttttaa 7380 tgactgcatagcattaaagt ggtagcaggt caaaaatacc ataatttagc tgggcatggt 7440 ggcaagtgcctgtagtccca gctattctgg aggatgagtt gggaggatcc cttgacccca 7500 ggagttaaaatccagcttag acaacatagc agaactctgt cttaaaaaaa aaaaaaaagc 7560 tagcaaaacacccctgtaat ttatttaact ctttttctat tttcagataa ttacattgtt 7620 tggttggttttttggctacg attcaataac atttaatatg taaagtatga ttcattttta 7680 ttaaacaaaactatgtatat atgcttgcct atatatgcat gaaataaaaa gctctaacta 7740 ttaacaacagttatccctag ggaatatagt attaggttgg cgcaaaagta attgcatttt 7800 tgccattaagagtaaggtta ccacctatgg gctttcgtct gtgggctaga tgagaaagaa 7860 agagggaagtttcactttta ccttattcac ttctatttga cttaaaacaa gcgtgcatta 7920 ttagagtaacttaaaaacta gcaataaaac actgtaacaa agtcttttgt atgagaactc 7980 ttctgtacccttttattatc ttctttggat aaatttctag aagaattagt caaaaatagg 8040 aacatttccctcatgcctgt aatcccagca ctttgggagg ctgaggcagc tagatcactt 8100 gaggccaggagttcgagagc agcctgggca acatggtgag accccatctc tactaaaaat 8160 acaaaaaattagccgggtat gatggtgctt gcctgtggtc tcagctactc aggaggctga 8220 ggtgggaggatcacttgagc tcagtgggca gaggctgcag tgaaccaaga tcatgccact 8280 gcactccagcctgggtgata gagcaagacc ttgtcttaaa aaaaaaaaaa aaaagatttc 8340 ttcagcaggatacagacccc ccacaaaaat gaacatttta aagattcata ttatatattg 8400 taaaactgccttcccagaaa tattttatca atttgtgtag ttttaccaga aataaatgag 8460 tgtccattttgctgctttct ggccaatagt agttattgac attcttttca tctttgccag 8520 tttcatacatggaatactat attacatttt gttttagctt ttattccttt tttttttttt 8580 ttgcaatggagtcttactct gtcacccagg ctggagtgca gtggtgtgat tttggcttac 8640 tgcagcctccatctcccagg ttcaagggat tctcctgcct cagcctcctg agtagctgag 8700 accacaggtgtgtgccacca cgcctggcta attttttgtg tttttagtag agacagggtt 8760 ttgctatgttggccaggctg gtcttgaact cctggcctca agtgatctgc ctgccttggc 8820 ttcccaaagagctgggatta caggcatgag ctaccacacc cagccaaatt ttgctttagt 8880 ttttattcctttgattactg catgagattg aatatttttt ctatcagcca tttttatttc 8940 tctttttttttcgagttgac tattcttgta ctttgctatt tttctgttgg ggtgtttgcc 9000 tttttaaaaattatttgcca tcaattttta tattataaat atatttgtca tatatggtac 9060 aaatattgtatcttatcctt ttgtttgtct tttaattttg tttataatat tcttttaaat 9120 aaatagtagttaggaatttt ttaagttgct aaatgtatcc agctggtagg agtaatttag 9180 ctgtttttgttttgaaactc ctatgtactg actatacaat ttaaattggg gcaggaaaca 9240 ctgaagcttagaggggttta aggaacttac tgaaggatcc ttcagctgag atgtagggaa 9300 gctagaattgagaatattaa tttttaagaa gttcttaagt ctaaatgaga atgagaaatc 9360 tggccaatgttgaagacctc taatgggtgg aggccccgtg gacatcagaa aagcggggca 9420 gtcaggggctggaagtcagg gtagaaatga caagtcagca aagcatcaag agtgaggaag 9480 aaaaagtagaaatgaggtgt ggccactggt actggcacca aaccccttgg caagtattgt 9540 ctataggtgaaagtagaaca agaaaataca cccaaatact tctaaaatga agtcatgcaa 9600 gacaatttttatttgaaaat gaaaaatgta gtcatcttaa tacaaaattt tactgacctg 9660 atttctgtgggatatgacac attttctttt tttagatttc atttgtttct tctcagcagt 9720 gattgctcctggaatgttgc atttttataa agaattcctt cgctactgaa agatagatat 9780 taaaatatggctccatatgg ctagataatg aacacggtac caccagtcca acttttaata 9840 tagcaaaacttcaccagaaa tatttatttt cttgatgatg gttgtcaaca aaccattgat 9900 gagatgtagggcactctgct aattctagaa atgttgtttc ctgccattga aagatcgttt 9960 tcaaagtgacattaaaagcc agtgaaatcc tagagaattt tagatggaaa tgagcagaaa 10020 gcatgttcttgaaaccaagt tagctttata gactactctg tctcttaatg taatttagat 10080 gcccatacaagaaaaagaag cttcaaagaa agaagtgatt cttctggaaa agatgaaaca 10140 tcccaacattgtagccttct tcaattcatt tcaaggtttg attttctaat attcgttaag 10200 tatttttataaagtataggc atgttgacat atgtaaaaag atttgttcct aaggactgtg 10260 tataaattaatttttgtaaa tgggtcattt ccccatttac ttaaattgca gcttgagacg 10320 tcctcgttatttcctctcta gtaagttttt gtagacggct ttcttatgtt ttcttgtttt 10380 tctgcctctccttaattctc actctcccaa aaaattaatg actggcttat tagcttcttt 10440 gctgtagtaacaacccccaa atttaagtga cttacaataa gacacatcta tttctcactt 10500 acattacatgctagctgtgg tgggctgggg tcttgagtct ggggcccagg ctgaaggagc 10560 agctcagataaggaaccagc tgttctcata agcaagagaa gaggggaaaa cacagagccc 10620 accacactatcgctctcaaa gccctgctag gatgtgtgtg tttgtgtgtg tgtgctgggg 10680 ggtactctgtttacatgaga tcctgcatat cctcaggcaa cagatgggac tgtgtaatcc 10740 tcttacagagagccagcaaa cagccacgca ccatagccta gcacactgcc acggagaggg 10800 ggagaactttagggaaggaa gtaccttcct ctgtacacct gaatacaatt ctgctgacaa 10860 ctttagggaaggaagtccct tcctccatac atctgaatac aattctgcct ccacgcatca 10920 ctgtagtccaaaggtaaaaa ataaataata aatgaaggag cattggtcag acagcattca 10980 ttcactgaactgatacttat tgagtgctta ctctatgcca ggcattgttc taggtgtcag 11040 gaatatagcagtgaacaaag cagatgaaaa tccctgtttt catgaaattt atattctagt 11100 gggaagagatagacaataaa caaatctaca gtatgtcagg tgtgtcttaa gttgtgacag 11160 ggctgtatgtgctgacagtt ttatgaaggg tcattcccca gcccagcccc cagcgcaggg 11220 ctgttttaagactgataatt agttcatgga gcagaagtgt taacctcaat atcttcaagc 11280 atcatcagttgggtaaaagt cagtcaataa ataaatacag ccactgtgtc ttgagtatgt 11340 aaactgtgcagagcactgtg ttccttactg attaaaaccg ctacattcaa ggtacttctg 11400 tgtgtatggcccttctttgg cttctgggta tttaaaaaga gctcttggga ctcttctgag 11460 gtcttcctgggagcagaaca gtacacatgg tctggaattg ggttgcatgg aataactttc 11520 aaggaaagccactgaataaa gtgccctgca ttcctgtcca ttggatactg ataatgctat 11580 aagatgatctttctcttctt tattttgttt gagattattg tgactctctg gctaactcct 11640 acttatcctcaggccttttc tgaactcaca attcaaatta cagctccctt tggttctctt 11700 ccacagcagttgtacttaca tatgtctatt tatataatta tgaatttgtt tcatatttgt 11760 cgccctttacatggtaaact taatgaattt tggggctcca tctgttttgc tcaccacttg 11820 atccttggcatgtagcacac aatggctgct caatacctat ttactgaatg agcaaatgga 11880 ctggaccacttttagagact ggagtatttc cttataccat gtgagattga tttttgagga 11940 cagtttaccactggaagctt ttgcagaact aaggtcattt ttacagtata cataacctct 12000 gctgtgtttgttgatactgt aagtttacat tttcttatga ctctttttaa gtagagcacc 12060 cctgtgtttaggaaagctag agctattgtg atgcctttga gtttgcttgg ctgattgctg 12120 ggacttgaactactgagctt atctaaaagc ctcagaggcc ttgtagcctc tgtcttttag 12180 agagtgtaggtaaaggcttg ttttccctca aatcgcttat ctctgatcat aagaaccatg 12240 gctctaatgtttgtctatag aaaatagaat gttttggccg ggcgcagtgg ctcatgcctg 12300 taatcccagcaccctgggag gccgaggcgg gcagatcacc tgaggtcagg agttcaagac 12360 cagcctggccatggtgaaac cccgtctcta ctaaaaatac aaaaacgttt agccgggcat 12420 ggtggtgtgcacctgtaatc ccagctactt gggaggctga ggcaggagaa tcgcttgaac 12480 ctgggaggcagaggttgcag tgagctgaga ttgcgccact gcactccagc ctgggcaaga 12540 agagtgaaactctgtctcaa aaaaaaaaaa agaaaatagg atgtttttat tggtttgaag 12600 caacataagaaaaataatga gaatgtagtg atattttcct aagacaaaat taattccatg 12660 tatattccatcaataaacat tcactaagtg tctgttatat gccaggcatg ttctaggtct 12720 tggagatatatcagcaaaca aaataggcaa aaattcccat gctgttgtat ttgttttcta 12780 ttactacataacaaatgaac acaaatttag tggcttaaca acaacaccta tttattatct 12840 cttgatttctgtaggtcaga agcctgaggt tggcttagct ggattctctg cccggagtct 12900 cagctagttgaaatcaaggt gtcagctggg actgttatct gtggctcatg gtcctcttct 12960 aagcttatttaggttgttat agatttcatt tacttgcaat tgggttaatt ggatcatggc 13020 tcactgcagccttgaactcc tggcctcaag tgatcctctc gccatggcct ccaaaagtgc 13080 tgtgagtactgtgcctggcc agaaagagct cttttacatt tatttaaaca cagagtttta 13140 ttttatattactctaatgca cacataaaaa agaaaatata agcaaacaaa gttggttaag 13200 gtattctaaaaattatttag gcagtgaaaa cattaagcct gccgggtcta cagcaagtga 13260 ttggaagatgccaatgtctg taagaaacaa tcttgatttt tttttttttt ttttttgaga 13320 cagtcttactctgttgccca ggctggagtg cagtggtgtg attacagctc actgcagcct 13380 tgaccttctgggcttaaggg atcctcccac ctcagtctcc tgagtactgg gactacaggc 13440 atgtacccccacacctggct aatttttgaa tattttttca ttatagagcc aggttttcgc 13500 catgttgcccaggctggtct caaacaccta agttcaagca atccacctgc cttagccttg 13560 gcctcccaaagtgctgggat tacaggagag agctgctgcg ccaggccctt gattttttaa 13620 aagtgcattttagaatgaat tataataatt gtttaataaa tgttggaatt tgacaaataa 13680 aaaggttatttagtgcccct caattgtttt gaagtgtcag tgatccatga gctttacagc 13740 agatggaaaatttgagagca taaatgattt ttccagacac ttccaataaa tataaaatta 13800 acagtggctaatgggggaaa atccttattt tacagtcaga taatgctaat tgacattaag 13860 tagtttcttttttttttttt tttttttttt tttgacggag tctcactctg tcacccaggc 13920 tggagtgcagtggcacaatc ttggctcact gcacctccac ctcccgggtt caagcaattc 13980 tcctgcctcagcctcctgag tagctgggat tacaggcgcc cgccaccatg cctgnnnnnn 14040 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnntaataa tcagggattt 14100 agaaataggatgaaatgaca agctaaagtc tttgagtctt agttcctcag ctgtaaagtg 14160 ggcatggtaccatttcacag gaattgatac gaggattaga agagattagg gctgtgaagt 14220 gcctgacacacagatagttc ttgaatcaaa tgagggtaaa ttgtagcact catctgtctc 14280 ataagcctaaactgcacatg tatctatata ttcatgacaa aaatcattcc aacacccagg 14340 ggctgtccaaaatacatctc tatctctctg tatacctatt ccctctccac cccccaaatc 14400 ctatacaaattcctcttcct cggccgggca cggtggctca cgcttgtaat cccagaactt 14460 tgggaggccaaggcgggtgg atcacgaggt caggagatcg agaccacagt gaaaccctgt 14520 ctgtactaaaaatacaaaaa attagccggg catggtggcg ggcgcctgta gtcccagcta 14580 ctcggagaggctgaggcagg agaatggcgt gaacccagga ggcggagctt gcagtgagcc 14640 gagattgcgccactgcactc cagcccaggt gacagagcga gattccgtct caaaaaaaaa 14700 aaaaaaggtatctctgtcat gatgaatttt tagtcagttt ctttcaccaa aagccagtgt 14760 aaggttgagtgagttggagg ttggtgaaaa ggaagtgaga tgaaataatg tgtctcctct 14820 gtttcattattctgattctt ccatggtttt gaaattgcca tcccttcatt gctgtggcag 14880 accttttactgactgagctt caatgacaag aaacatcaat tctcctaaaa gtaacacatt 14940 cctgaaaataaccgatctct agacattaag gcatatgggg aagcatcttc tcagaaccct 15000 tcctagatctccagtcttac ttgcatgcgc tttctcctca ccctccagcc tacagacttc 15060 ttcacagcacttctcaccca atgctgaggg actccccaac tagacttcgc ctccttaaca 15120 gtagaatggatggcttcttt gctttttatt cctacacagc attccttgct tttgcatgtc 15180 ctcaatagaagtttgttaat aactgaatgg atcgtctctt aaagaagaga ggaggaaaaa 15240 ttgaaatatgtgaaagaaga tgcatggttt gtgaattaga agcaaccaag ggtagacact 15300 gccaggttactgatatccac agtaaagttg gttagggtac tttaaagagt aggatagcaa 15360 aagatagatatttggcaaga gattttggca tttaatgggt actacagggg aaaatgttat 15420 caacaattgcttataagact gattttggcg cttatgtttt gtgttccttc agggtttttt 15480 tgtttgtttgtttttaatga atccactcaa caaacattta agcccctttg atgtgctaac 15540 tactgtttaggtacaaaaga atgaagtgta gacaaacaag tgagtagaaa tccttctttt 15600 ctaacaagatcccagctgtt agttggttgg ctaatgaaga aagctggtta gagcagaaaa 15660 tcatctgttttagtctattc cagcagctat aacaaaatac cataaactag gtggcttata 15720 aacagcagaaatttatttct ggcagttctg gaggctggga agtgcaagat caaggtgcag 15780 gcagattcagtgtctggtga gggttcactt tctggttcat agatggtccg cctcattgtg 15840 acttcacatggtggaagggg agagggttct ctcttggaca gcaatccatt aatgtgggct 15900 ccaccctcatgacctagtca ccttccaaaa gccctatacc tcctaatacc atcactgtgg 15960 gggttaaaatttcaacatat acatttgggg aagacaaaga catttggatc ctagcaatta 16020 tacagacatattttaacata agaagacata atcatccttt gagtggaaat ggccaggaaa 16080 aaaaaaaagaaaaaaaattt aaggaaatga caagcatttg ttaaaggata atttcttttc 16140 tttaatacggagcaagtgtt tgtggataat ctgtccacaa tccttttaga agttttctag 16200 ttatatttcattcatttcat tcaacattta ggtcaatggt tatttattta tttattttta 16260 aactcactgagtcctccaaa atattcagca tagcttttgg aggaataatc acatctttca 16320 ttttctattcatatttcatc agtttatgta ataaagacaa gaataactca ctacagttca 16380 agaaaattcagaattatagt tggtagatta tgagtccact gactatagtt ctgaattttc 16440 tttcttatgtaagttatgtg tcttatttag aatttctagt ctcttttctt taatgtggag 16500 caaggatttgtggatagtct gtacataatc cttttagaag ttttccagtt atatttcatt 16560 catcccatcaacatttaggt caatagctat tttttttaaa aactactcac ttttactgag 16620 tcctccaaaatattcagcat agcttttgga aaataatcac ctttcatttt ctattcatat 16680 ttcatcagtttatgttacaa agacaagaat aaatggcgta aacatatttg ggaaaaaaca 16740 aaatgatcttggtaagattc agttcaattg gtaagagcag aggtacttgg acatactaga 16800 gagcctagtagtatttagtg gtaacgttga tggggcaata gcaatggaga gtgtcctgta 16860 atctagtgagtggtttaggc agaggtcagt tataagagtt tctattgtat tcaacaacat 16920 agataaataagtgccatata aatatggctt tatgcccaaa ttcaagaagg ggctattaat 16980 tcttcctggggtgttatgaa agagtcacag ctgctatttt tacagcagat ttctaacctc 17040 tcaaaggaatgtattaataa aaatagccaa catttcttaa actctcatta tgtacaggca 17100 ttattctaagcatctgacat ggattaactc atttaatctt tccaacaatc caaagaagaa 17160 atttctataatatttccatt ttacagatga ggtaatgagg cacggagaag ttaaagtgac 17220 ttgcccagagtcacagagct aagaaatata aatggtatag tggaattaga acccagtcca 17280 tcttgattccacagccagaa catggctgat aaatatctga aatccttcca gcaccaaatg 17340 attccttttcttacagagaa tggcaggctg tttattgtaa tggaatattg tgatggaggg 17400 gatctcatgaaaaggatcaa tagacaacgg ggtgtgttat ttagtgaaga tcaggtaaaa 17460 acttctaatttgctttttaa ttttatgtat gtgtggtggg gggagaatta aatttataat 17520 gtcataagtagtagactatg ttgatataaa gcatgtgttt tggtagacag attgaaacca 17580 taaaatagtggatcaatatt cttggtagat tcagtccaga acaagtttgc aattgaactt 17640 aaactgaattgttttttaga gagtgagatt ttcttgagaa aagataatct gtttggaaaa 17700 tctcatgtaggatgcctctg agataagtct tcatggttaa aaaaaaatct gaatgtgagt 17760 gttccttagccatttaacat gtaacatatt tatagcttca ctgttttctc ttttaccatt 17820 tggtgctctgttttaaacta gatatcccac tttgctaggg aggatagaag cttgtcttgg 17880 caatgcctatttagtttcat tggttattaa gagggggaga taaaagatga agaataatgg 17940 cctctcccactttctctttg cacaaatgta tttctcttct atactccaag cctccctgga 18000 actctcttgggagtggtact catgaaggag acaggtttgt atgtgtggag aggaatctga 18060 attagctcctttatgattga tggtaaggcc attgcctcaa gaagcacaca ggaaaggccc 18120 accatctttcctttggccat tgtttctttg ttctatttta gcatgtaaga gcatcatgcc 18180 catataatttcctcttaaag ttgaatattt tgaatatatg aaggattaaa aaatcaatat 18240 ctctaacttctgtaagatta atcaagcatt ctttgtatgc tcatttatat tatatattaa 18300 attccatattgatagaaact cttttttctt atctaggtat tattttgcca cattttataa 18360 aaatgcttctcatgacaaaa ttttgagtta catttctttt tgttgggaat gaactaaaat 18420 tacaactgaatattaatgtc tggaatatag ctttattcct attattttcc tctttctgta 18480 taatttggcagacaaagaac cagtgaaatt ttagaatagt ttaaataaat ctctgtaggc 18540 atagaacacattttcataaa gaggctcatg gtcaacaaag ataaaatcaa atcatgactt 18600 agaaataaaactaaacttca aaggtaaagt atttgttggt tttatattag atatactgat 18660 attttattacaattcctaac ctcacagatc ccccatttct tcctctttct ctccccaccc 18720 ttgtcaccctccttccactg taaaggaaga accaatggct cccaggttat caggaaacag 18780 ggctgcttgtgtactattca cgatgcagtt agcaccccag ggttaagtag gaaaaaaaga 18840 aaaacatgaacggcatgcct ctttcccttg cttctactta tctttttctg catgtggaat 18900 ttcccttgattttaccagtg atatttggat tacttttctg tgcctccatt tttttagttg 18960 tagaatgaaaataataatat gataaagtgt acctattaac ttcattccta taaatacaca 19020 tacactatatgtgtgtatgt gtgtgtgtgt gtgtgtataa tttctatctt tttgcatgtt 19080 accatgaagacatttcagtg actaccaggc tattcagtgg ctttgttttg tgttctctct 19140 atagatcctcggttggtttg tacagatttc tctaggacta aaacatattc atgacaggaa 19200 gatattacacagggacataa aagctcaggt aacagctcag agagaagact aagacagaac 19260 tgatcttttcttgaagtacc tcaaacaaca tgacattttc tccatttata gaacattttt 19320 cttagcaagaacggaatggt ggcaaagctt ggggactttg gtatagcaag agtcctgaat 19380 aagtaagtactttgaaaata atttttcttt ctagtcaaaa tagcccaaat atgtattttt 19440 agatatcatggattaagaag atattaaaat cttggttgtc taaataattt taggtagctt 19500 tatgtaaatgcattacatca gatggtactt tgagattaaa attctcaaga taaattgtgg 19560 tgtaatagaatgatgttgct aatattctgt agtgtgattc cagtttgtca aatatggatg 19620 tgactgtaatatgcataaag ctagagagaa tttcgtgaaa taggcaggtt tacacttctt 19680 aatgaaaaaagtcaaactct ataaaatatt tgaagagatt tattctgagc caaatacgag 19740 tgaccaaaggtccatgcctg tgacatagcc ctcaggagat cctaagaaca tgtacccaag 19800 gtggccggtctacaacctgg ttttgtacat tttagggaga tgcaagacat caattagatg 19860 tacatgggtttggtccagaa aagcaggaca actcaaagct gggaagaatg ggagggagct 19920 tccaggtcataggtggatta aaaacttttc tgattggcaa ttgattgaaa gagtctatct 19980 gaagacctggaattagtgga agggagtgtc tgggttaaga taaggggttg tggaaatgaa 20040 ggtttttattatgcagatga aatctccaag tagcaggcct cagagagaat agattgtaaa 20100 tatttcctcttatcggattt aaaaaggtgc cagactctta gttaactttt tcctggatca 20160 ggaaaaagccttggaaaaag aagggaattt tcttcagaat gtagattttc cccacaagag 20220 atacctttgcaggactattt caagatatgg acaaagaaac atgatttggg gtaaaatatt 20280 ttgattcctttcaggcctgc tatctgtcat gtgatgttat actagagtca ggctggactt 20340 tggtatcttattgctacaag gagtctgctt tgtcagtctt aaggtctgtt ttaatgttaa 20400 tgctggtcaactgtgcctga attccaaagg ggaggaggag ttaatgaggc atatcagacc 20460 ctgcttcccatcatggcctg aactagtttt tcaggttaac tttggaatgt ccttggccaa 20520 agggagggtttatgagttgg ttggggggct tagaatttta tttttggttt acacactttc 20580 tagcaaaataaatttgtgca cctgtttgga agacaatttg gtggcaatat gtaccaagag 20640 atttttaaatatcctgtttc tgggacttct tccaagggaa taatttgaaa tttggaataa 20700 cgtaaatgcctaaataattg ggaaatggtt aaatttaata aagcttggca tggccatggc 20760 catgtacctgaatatatcat aaacatttat ggttttgaag acttcttgat aactttgtta 20820 tactaagcaaagaaaatgga attctgaatt ttaaatacat tgtgatcacg gttatatgaa 20880 aaatatgtgtggaaagaaga caggaaggaa atatatcaga attttaacaa tagttgtttt 20940 aggtgctaagattctgggta acttttttct cccttattca tttttgtatt ttccaagttt 21000 taaatcatgaggttgcaatt tgataatctc tacatctgag agatttttat aacatgacaa 21060 tttcatctctttgtggagtc tttaagccat aaaaaatata ttttaatgtg taaatttttg 21120 ggaggtgaattgtaagttta aaaatcagct gatttagtta ctttatcaac atacagtgtt 21180 ttgctttcttctaacacatg tatgcatcaa atcttgtgtt atccattttc acattttttc 21240 ttgcatgtccatgtcttaag acttttctta ctccaataaa aaatcatgct gatttattat 21300 ttaatataatttactagttc catggaactt gctcgaactt gtattggaac accttactac 21360 ctgtccccagagatctgtca gaataaaccc tacaacaata aaacgtaagt tgctgactct 21420 tagtttgaaagtgtcagtaa aatctgatgg atgacactga atgaagattc cagaaactaa 21480 aattcaaatctcttctttct ttcttatggt acttttgtaa tttcatttgc ttcatgtgtg 21540 aaattgttctggaccaaact gagggttggg ttgctatttc tcgcggtcca atacgagatg 21600 cagatgaactggggaggaag agagttttta tttctgtaac cagtacaggg agaaggcctg 21660 gaaattatcaccagaccgac tcaaaattac aaagtttttc agagcttata caccttctaa 21720 gctatatgtctatgtgtaag tgtgcattca tttaaagaca tactgattaa ctccttttaa 21780 tctataactaaggtctgagt cctgaagact ttcttctgga gcctcagtaa gcttacttaa 21840 tctaaatgggtctaggtcct ggggtgatta cccttatttt gtctcctgct aaatcatgga 21900 ggtttagggagttcctgcag acctccaata aacttgtttg tggaggcctg gggagtttct 21960 tcagaccaccaataaaactt gtttaatctt aaaaggctcc ttgttaagaa ttccttcatt 22020 attttgtcatggtttaaggc ccaggaaagg cctaggcaaa actcttggtg ggcttttgtt 22080 acattacagcctttgtataa gggcactggc tttttttttt ttattttttg agatagagtc 22140 ttgctcttgtcacccaggct agagtgcaat ggcacgatct cggctcactg caacctccac 22200 ctcccaggttcaagcgattc tcctgcctca gcctcctgag tatctgggat tacaggtggc 22260 tgccaccatgcccagctaaa gttttgtgtt tttagtagag atggagtttc accatgttgt 22320 ccaggctggtctcaaactcc tgacctcacg atctgcctgc ctcagcctcc caaagtgttg 22380 agattacaggtgtgagccac tgtgcctggc tgggcactgg ctttttaagc ttttaatatt 22440 taacttcaccactcagttag tatagaaaca gttgtgatgg aggcctgcat tggtaagacc 22500 tggcctgccacaaaatgggg atcccagtga ctatctctga gcagtgttac ctgaaggttt 22560 caaacttgtttagaagaaag ccatttctct tcatttaaag atacaagtgg tataaaaaat 22620 aacatcgaaaattgcagtca ctgtgatgtc catttttgta ttatatgttc atatctttga 22680 agcactgtttagtctattgc aagaaagatt gaagaggatg aagtagaaga caatgtggtc 22740 tggtgaccgctcactggatt aggagctagg aatcctagtc ttggctcagt tgctaacttg 22800 accaagtcagttgacctctg tgggcttcag ttccctaact cataataatg agagtattga 22860 ctaggtaatcttcaaggtgt cttccagctt taaaacccag ttagttttta tgtatgtgat 22920 atcagagtctggttctcagc aataattttt tttttttttg agatggagtc tggctctgtc 22980 atccaggctggagtgcagtg gtgtgatctc ggctcactgc aatctctgcc tcccaggttc 23040 aagcaattctcgtgcctcag tctcccaagt acctgggact gcaagcacgc cccaccatgc 23100 ccagctaatattttgtattt ttagtagaga tggggtttca ccatgttggt cgggctggtc 23160 ttgaattcttgacctcaggt gatctgcccg cctcagcctc tcaaagtgct gggattgcag 23220 gtgtgagccaccgcacctgg ccctcagcag taatgctaat gtatactgca agaaaaggtg 23280 aagaggagcttttgcttcct ataaggagaa ggaaaaaaat ttcatttttc aaagctggct 23340 gccattgaacaagttggcga taaggaagat tgagttccct ttggaagtta attgtccttt 23400 tgtttaggaaaaaatgccca agagatactt ggctattgga ctttgaagga gataaatgga 23460 aggcaaagctcagacaatag agatttacaa aaagaatagt aagaatttct ctgcataata 23520 aaataacagggatttttttt ttttttgaga catcccctgg caccaaggag tttggcctca 23580 agttagttgtgcaggaattc aggtagggtg tgttggacgg aaagtaggct gttcagagca 23640 gggcatgccacagacagcct tgggtcagct gcattgtttt gtttgcttgt actgcttttc 23700 aagaatttgaatcaacattt aaatgctgct ggatatggtg gctcatgcct ataatcccag 23760 cactttgggaggctgagggg gatgattgct tgagtccagg ggttcaagac caacctggac 23820 aacattgtgagaccctgtca ctacatccaa aaaaaaatta aatattgaaa gactttaaaa 23880 tatgcatagtttgtacctct gaaaattgga agatcttagc aataatcagg tgggtagccg 23940 ctggctccattagaggactg gttcaccaca gtcctcaata tgcagagtgg tctcaggcct 24000 gcaactggccccacccaacc cccaggtggc tgcagtactg cctgagccct gggggcatat 24060 gaattctctgccctggctgc agagggtcct ctgggaacag aagagaagtt tgggtctgtg 24120 gaagccctagtaaagacaaa agtctgtgtg gtgtgaaatg gtcagtgagt ttctagaagg 24180 tctagaaagttcatgtttgt ttcctgggtc aggtgcaggc ggctcacacc tgtaatccca 24240 gcactttgggaggccaagaa gggagtattg cttgagctca agagtttgag accagcctga 24300 gcaacatggtgaaaccttgt taatgaaaaa aaaaattatt aaaaaaaatc ccacaaattt 24360 gtttcccaccaatcttaccg tctattgtac ttactaccat cttttgtact caaactttta 24420 gtatgagtctatctctctct ccttctctct gacacacaca cacacacaca cacacacaca 24480 cacacactcatgcacaaagc attgctgcta gaggagccat ttacctcact cctcacttta 24540 atgattccttcttgctttga ctccttgact tctgattaga cattttttga tcttttagat 24600 ttaattgtgctttttgttct ataaaataac tcctcaaacc aatcacatat aaatatttat 24660 gaagtactaaatctgtaagg agcaaagctc atgatatata ttttaagtat atttttaaat 24720 gtttattgagaatcagatac tatgtttatc acataatata actttggttc tgtcaaaagc 24780 cttgagtaggatatatcttt caaaatcaac caaatattac cttttgagtc aaaacaaatc 24840 catgtttgagttctgcctgc ctcctccaaa ttgctcaaca tttcatcata catacattgt 24900 ttttgagcaggaagctgaac taaatattaa gccaccaggt tgtagcaaag tttgtgtgcc 24960 tttctttgactagaaatctg acaaactaca aatggttttc attttacctc ttatcttcta 25020 ataagaattgatgatatatc tgaaagcatt tgtaaaagct gatcaactta cataaaattg 25080 taaagcgacacaaatttaag gcactgtaag gataaaagct tttattaaga attatggata 25140 ttttcttggcatgtaaactc ttatcttctt tagggatatt tggtctcttg gctgtgtctt 25200 atatgagctctgcacactta aacatcctgt aagtatgctc attgtcagac taatcttgaa 25260 ttattggaattgtagaaaag aaattaactt ctgggagaaa aaggttaatg tttggtttta 25320 ttagattgttaaaaattata tggataagct acttaaaata atgatagatg acatggaaag 25380 ctgtccaagcaatattataa agtaaaaagt ccaagttgga gaatagtatg tgtagcatat 25440 ttccattaaaaataaattgt gtgggcttgg cgtggtggct catgcctgta atcccagcac 25500 tttgggaggctgaggcgggt ggatcacttg aggtcaggag ttggagacca acctggccaa 25560 catgatggtgacaccccgtc tctactaaaa atacaaaaat tagccaggca tggtggcatg 25620 tgcctgcagtcccagctagt tgggaggctg aggcacgaga attgctagaa cccaggaggc 25680 agaggctgcagtcagctgag attgcgccac tgcactccag cctgggtgac agcgagactc 25740 catctaaaaaaaataattaa ttaattaatt actgtatgaa tagatacgtt cagcaaaaga 25800 aaaatgtacatgggcaaagt tcataggaaa ccaggcacaa gcttttaaga gtcttttccc 25860 agaggtcacatgggatgtgc caaatcctcc agcattgtta cccacgtcac ctgtgaaatg 25920 tgatctataagaaagctcat cggatatacc cagtgcccag gatttttact ggggactggt 25980 cacataggcaccctctacct ggcatatgcc aaacttccag actcctggaa agaaagcccg 26040 tgttcagcataaaccatttt gttcacataa atagctgagg caaagatagc cactcttgac 26100 attcagggaatggtgggaat tcttctgaaa tcttagttcc cagacaccag ccacgggcca 26160 acattgtaagcaggcctttc tgaggagagc ttgctacatc aactcttttc tccacagctg 26220 tcatcattgttattaattat tgtcaagggt tgcacagcca gtgtctgacc aaaatgtgta 26280 ctccattgtttttttgagat ggagtcccgc tctgttgccc agactggagt gcggtggcac 26340 gatctcagctcactgcaacc tctgactcct gggtacaagc aattctcttg cctcagcctc 26400 ccgaggagctgggattacag gcacccacca ccacacccgg ctaatttttt tgtattttta 26460 gtagagtcagggttttgcca tgttggccag gttggtcttg aactcctgac cttgggtgat 26520 ctgcccaccttggcctccca gagtgctggg attacaggcg tgagccacca tgcccggcca 26580 atgtgtacctttattgctac accatggagt tgaatattat tatgtataaa taactattgg 26640 tttcatacaatagaagattt ctggtctatg aagcatttta gaggaaatta aacgatgttt 26700 atgttaattttaaaaagcaa gagataaaat ttcatatcaa tatgacctca actttgtaaa 26760 ataaacatcatttttaaaag agatcagaag gagctatacc tctgagtggt aaaattatac 26820 atattttcccctgtctttat aacttcctat accttccagt ttttttatta tgagtaaaca 26880 ttattttgataataagacag aattaaaaca aaataaaaac ttgttttaaa taacatggca 26940 tcttgttgaataactgcagt atctgctcat gaaagattag ttgatgaaaa caatttaagg 27000 tggaccacagtgcttctttt ttattttttg attgagacag ggtctcactc tgtcacccag 27060 gctggagtgcagtgacgcaa tcacggctta ctgcagcttt gaccgcctgg gcttagacaa 27120 tcctcttgcctcagcctccc aagtagctgg gaccacaggc tcatgccacc aagcccagca 27180 aatgtttaaaaaccatgatt tggagagatg aggtctaact atgtttccca ggctggtctt 27240 gaactcctgggctcaagtga tcctcctgcc ttggcctccc aaattgctgg gattacaggt 27300 gaccctagtgcttctaacta caatttaaaa acattgtttt gcttcttggt atatttgtta 27360 ctttaacacttttattattt gttactttag taacttttct ctgatttagt gtcatttctc 27420 cttgtcctttcagtttgagg gtaacaactt acagcagctg gttctgaaga tttgtcaagc 27480 acattttgccccaatatctc cggggttttc tcgtgagctc cattccttga tatctcagct 27540 ctttcaagtatctcctcgag accgaccatc cataaattcc attttgaaaa ggcccttttt 27600 agagaatcttattcccaaat atttgactcc tgaggtaagt tttgaggtga ctgtttggat 27660 tttggcagagattttgggtt gcaggtcctt gacacgtgtg ttcggtttta ggtcattcag 27720 gaagaattcagtcacatgct tatatgcaga gcaggagcgc cagcttctcg acatgctggg 27780 aaggtggtccagagtaagtg tgactttggc atgcaatcaa aagtatttat tacacatgtc 27840 tcacacagagagtaatgcaa ggaaatttca ccaaacatat tgaaagtgga cattttaaaa 27900 aatacaagcagtataagcag gagaaaaatc atcttgtcaa atggcaacta gtgagtgtgc 27960 ctgaaagttgtatatctagc tcatgcatga cctgcagggt tccttctcgt tagtcaggaa 28020 acctccatgaagcagaggac atgctaatag agatgcttga agaggttgag cccaaactta 28080 acttttgtgtagtgaaggga cagagtggga gaaggttgca gatagacatg gatgatgaga 28140 tgaaacttatttttctaaaa gaggatagac tggcaattaa gaattctgtt gcaaaggacc 28200 attggagctgaagttaggat cttggggcct aattgataac agtaagaact gttactttgt 28260 ggttcccaaagaaggcagga gatattttat ggtagtaata aatacagaaa actttttttt 28320 ttttccgagacggagtctcg ctctgtcgcc caggctggag tgcaatggcg cgatctctgc 28380 tcactgcaaactccacctcc cgggttcatg ccattctcct gcctcagcct cccgagtagc 28440 tgggactacagccgcccatc accactcccg gctaattttt tgtatttttt tagtagagac 28500 gaggtttcactgtgttagct aggatggtct cgatctccgg acctcgtgat ccgcccgcct 28560 ctgcctcccaaagtgctggg attacaggcg tgagccaccg cgccaggccg gagaaaacta 28620 ttttagtcctggtgtcaaga atcagctaag ctgtgtgtca gagggagggg tacgttaaga 28680 aagagaaaattactaattca tttgatgctg tgaaagtcaa agccccagaa tttagctgta 28740 actgaatgcctggacttaca atatcaggag gagcagaaag cctctcaaag gaatccatga 28800 cagggaaatgttatccattg agacagagat tctaaaatca aggaaagtta aagagaaagt 28860 gaatgagcctctttgccatt taatttgact aacattgttg tataccagtc tagattgaga 28920 atgtttagaaaatagacaag tacagagtat gggactgtgt attgtccata tttctaatct 28980 aggtaagataggagaacaag aacaattttt tttttattga gatggggtct cactgtgttg 29040 cccaggctggtctcgaactc ctgagctcaa acaatcctcc taccttggcc tcccaaattg 29100 ctgggattacaggtgcgagc caccttactc agcccaagaa caaattttga tggagataaa 29160 gacaagcattagaagatcta ctcatacctc agtcctggca ctttgggagg ccaaggaggg 29220 caggtcaccggaggccagga gtttgatgcc agtctggcca acatggccaa accatgtctt 29280 tactaaaaatacaaaaatta gctggacctg gtggcccatg cctgtaatcc cagctccttg 29340 ggtggctgaggcacaagaat cgcatgaact cgggaggtga aggttgcagt gagctcagac 29400 cctgccactgcaccgtagcc cgggtgacag agtgagactg tctcaacaaa aaaaaaagag 29460 agaagatctactcataaatt ccaaacaatg tggcatgaat ggagtggcct gataacccaa 29520 gctctaatgaccaaatttaa taacttttat tattacccca tacatattgt ttctgtaaat 29580 gttaatattaatttctattt ttctgaaaaa aagtgatgtt atatattact agaaatatgc 29640 aaagggactctgaaaaaatg gtttttttca tttaaagaaa ttgcatatta atttttcatc 29700 agtactctcactgtgtgtaa aatatctctg gctaaaaagt aaacttactg tgttatgaaa 29760 tgtagcttatgtttatactc ttacaagtat cagtattaat ggtgtacaat ttttaaaaaa 29820 ttgaagctgttttattttgg ttaattaaga gtgtaaaata caaaaagtga gattccaggg 29880 aaagtgcccaccaagatcaa ggatatctgt gccaattaaa aggaatgcta tattgcatag 29940 aaatgaatggagaccaccag ctggagccca gaaggccaga tctgtaagtc attctaaacc 30000 ctcctttgtgttttttagct atggtatatg ctttttgttt gtttgtttgt ttgttttgag 30060 acggagtctcgctctgtcgc caggctggag tgcagtggcg cgatctcggc tcaccgcaaa 30120 ctccacctcccgggttcaag caattcttct gccccagcct cctgagtagc tgggactaca 30180 gacgtgtgccactatgccca gctaattttt gtatttttgg tagagatggg gtttcaccat 30240 attggccagaatggtctcca tctcttgacc tcgtgatcca cctgcctggg cctcccaaag 30300 tgctgggattacaggtgtga gccatggcgc ccggccccgg ctaattttta tacttttagt 30360 agagacagggtttcaccatg ttggtcagac tggtctcgaa ctcctgacct tgcgatcagc 30420 ctgcctcggcctcccaaagt gctggtatta caagcataag ccactgcacc cagctgttat 30480 attctttttctttaattttt taattaaaaa aaaaattttt gtgggtacat agtaagtgta 30540 tatatttatggggtatatga gatgttttga tacaggcaag caatgtgaaa taagcacatc 30600 atggagaatagggtgtttgt cccctcaagt atttatcctt tgagttacaa acaacccagt 30660 tatactctgtaacttatttc aaaatgtaca attaagttac tattgaccat aggcagtcta 30720 ttgtgctatcaaatagtagg tcttattcat tcttttgttt ttttaaccca ttaagctatg 30780 gtatattctgacagacctat ctgcacatgt tcatgaggta caagcttatt gtttggagtc 30840 cacaaattttgtacttaaaa tgaagtattc tgtactgagc attataatgg tattttgttg 30900 gacaacttctagtttttata ttttatgaaa caatgctgta tgctcttata agtatacttt 30960 aggcttaattttctttttat aactgaaatt cttctaattt ctaataaata agatttttct 31020 gtataggaaaagtgagtaac atagcaacag aaaacactct gcatttaata ttcttaattc 31080 taacatattatgtataggat tgagaagttt ttatgatata ataattgata tttccctagt 31140 gattctttgtgtttaattat ttgaattcac ttcagcagag tgttgaatct tttaggtcat 31200 actagtgaaatgcttctggt atgtaaatga taaaatggct actgtctttt aattaaagaa 31260 ttgtatttttaaagaaggct catggttaaa ttaagaacca tttggaagtg tatttactaa 31320 gtgtttacttgatatataga cattttagaa aatgtgttgg tatataaaca tttttttaaa 31380 aaccgattgtttaagttatt gcccttcatt tgataaaggg ctttatttat ttatttattt 31440 atttatttatttatttattt atttatttga aagagggtcc tgctgtgtca cccaggctag 31500 agggcagtggcatgtctcag ctcactgcag cctggatgta ttagtctgtt ctcatactac 31560 tataaagaactgcttgagac tgggtagttg ataaagacaa gaggtttaat tggcttacag 31620 ttctgcaggctgtacaggat gcattgctgg ggaggccgca ggaaacttat aatcatggca 31680 gaaggggaagcaggctcatc ttaaatggcc agagcaggag aaagagagca aagggggagg 31740 tgctacacacttgtaaacaa ccagatctct ggagaactta ctatcacaag aacagtaaga 31800 gggaaatctgtccccataat ctaatcacct tccaccaggc ccctcctcca acatcaggga 31860 ttacaattcaacatgaaatt tgggcaggga cacaaatcca aaccatatca ttccaccttt 31920 ggcccctcccaattcccata tccttctcac attgcaaaat acaattatcc cttctcaaca 31980 gtcccccaaggcttaactca tttcagcatt aactcaaaag tccacaattc aaggtctctc 32040 tgagacaagtcaagtccctt ccacctgtga ggctgtaaaa taaaaaacaa gttagttact 32100 tccaaaatacaatgagggta caggcattgg gtaaatacac ccatttcaaa agggagaaat 32160 cagccaaaacaaagggttta tagaccccat gcaaattcaa aacctagcag ggcagtcatt 32220 aaatcttaaagctccaaatt cctttgaccc catgtctcac atccagggca tactggtgtg 32280 aggagtgggctctcaaggcc ttgggcagct ctgctcctga ggctttgcag gctacagccc 32340 ctgcggctgctctcacaggc tgctgttgag tgtctgcggc ttttccaggt gcgtggtgca 32400 agctgtcgttcaatctaccg tttttggagt caggagaatg gtggccctct tctcacagct 32460 ccactaagcagtgccccagt ggggactctg tgtggaggct ccaatgccac atttcccctc 32520 tgcactgccctagtagaggg tccccctgaa acaggcttct gcctggacga ctaggctttt 32580 ccatacatcttctgagatct tggtggaggc tcccacgcct caactcttgc actctgtgca 32640 tctgcagacttaacaccatg tggaagccac caagatttac ggcttgcacc ctctgaagca 32700 atggcctgagctgtaccttg ggccgtttta accatggctg gagctggagc agccacaata 32760 caggacaccatgtcctgagg ctgcacagag cagtggggcc ctgggcttgg tcctcaaagc 32820 cattcttccctcctaggcct ctgggcctgt gatgagaggg gctgcctcaa aggtctctga 32880 aatgccttcaaggcatttcc cccattatct tggctaacaa catttgactc ctctttattt 32940 ttgaaaatttctgcagctgg tttgaattgc tccccagaaa atgggttttt ctttctaggc 33000 tgcaaactttcctaactttt acactctgct tctcttttaa gtataagctc tggttttaca 33060 tcatttatttgctcacaaat atgaccatag ggtgctagag cagccaggcc acatcttgaa 33120 tactttgttgcttagaaatt ttttctgtca gacgccttaa atcatcactc tcaaagttca 33180 aagttccacagatcccctag ggtagtggca caatgcctcc aacctctttg ctaattcata 33240 acaaaagtgtcctttgctgc atttctcaat aagttcctca tctccatctg agacctcctt 33300 agcctggactttattgacca tatcactatc agcattttgg tcactatgat tttaagaagt 33360 ctctagggcattccaaactt tccatcatct tcctatcttc ttctgagccc tccacgctct 33420 tccaacctccgcccattacc cagttccaaa gtcactttca cattttcagg tatctttata 33480 caataccccactcctggtat caatgtactg tgttagtcca ttctcatact gctataaaga 33540 acacctgagactgggtaatt tataaagaaa atacatttaa ttggctcaca gttctgcagg 33600 ctgtacaggaagtatggctg gggaggcctc aggaaattta taatcatagc agaaggggag 33660 gcaggctcatcttacatgca ggaggaaaag agtgaagggg tagccgctac aaacttttga 33720 acaaccagatctcatgaaaa ctcactcact atcacaagaa cagcaagggg ggaatctgcc 33780 ccaacgatccatttaccagg cctcgtctcc caacattggg gattacagtg caacatgaga 33840 ttgggcagagacacaaatcc aaagcatatc actcgacctc ccaggctgag acacaaatcc 33900 aaagcatatcactcgacctc ccaggctcaa gtgatcctac cgtctcagcc tcctgaatag 33960 ctatactaccggtatgcacc atgatgccca gctagttttt actttttgta gagtcagggt 34020 ctcactgtgttgcccaggct gttcttgaat tcctgggctc tagtgatatg cccgcctcag 34080 cctcccaaagtgctgggatt ataggcgtga gccactgtgc ccagcctaag ggcttaattt 34140 tattaaagaaataagaaaag tatgttgtga ttcagaggac tctttatcag acctgtagaa 34200 gggaaaacacatctaaaaga tttgaggatg aattaaatta cgaactgttg aacacgctga 34260 catttttccagttccttgaa aaggtaaaat tgatttccac aggaactacc tctgatattc 34320 ctattactgttgggatgtta gagaacattt taaagaaaat gtttattgcc tttcaatact 34380 tttctatattttttaccact tttcaacaag tcattagtag cattttcttc taggttgtat 34440 ataggtgaaattgtaaaaca aagaaaacta cttcttgttt taaaagattt taaaaatagg 34500 caggtgcagtggctcacgcc tataatccaa cactttggga ggctgaggca ggaggatcat 34560 ttcagcccaggagttcgaga ccagcctggt caacacattg agaccccacc tctacaaaaa 34620 gtaaaattaaaaaaaaaatt ttttgttttt tactggacac agtagcatgt gcctgtagtc 34680 ccagttacttgggaggatga ggcaggagat ccctggatcc caggagtttg aagctgcgat 34740 gagctatgatcacaccacag tcctgcaggc tgggtgacag agtgagatcc tgtctcagaa 34800 tttaaaaagaaaagaaaata ttttaaaaat aaacatataa tttgtattta gattaatgaa 34860 ctaaattttatacatttact taaatattta aatagaacta tatgaaagtg ccatttttct 34920 agattaattatggtcaattc tgggcaattt ctttttttga gacggagtct cactctgtca 34980 cccaggctggagtgcagtgg tacgatcttg gctcactgca agctccgcct cccgggtttg 35040 tgccattctcccgagtagct gggactatag gcacccgcca tcacgcccag ctaattttgt 35100 ttttgtgtttttagtagaga cggggtttca ccttgttagc catggtggtc tcgatctcct 35160 gacctcgtgatccgcctgcc tcagcctccc aaagttctgg gattacaggc atgagccact 35220 gcgcccagccaattatgtgc aatttcatat ggtccaatct aacatatatg tgaaccatat 35280 agcagtaaaaacaacaaaga atataacatg ttacctcttt acatgaggac attttggttt 35340 taattgttcttgttattcat attcccaact attagttcct aggtctttcc agtagtttta 35400 tctttttttctctttttatt attaactgta aactgtaaac tagacagagt tgccacgctt 35460 taggttaaattgaccccact ttgctcttta gcaagaaggt cttgactggc ttttatatct 35520 taatttgatctgtttcttgt cttctagctc agtggcttct actcagttgg aagataaact 35580 gtcatttctggttctcctat tctcattctg ttctggttgg gaagggtggt gagggctggg 35640 atggtgatatgcccatcatg gctgttatat gacctttttt aatattttct ctggaagaat 35700 gattctgattcagcatcttc tttcctttaa gtcatgatgc cattttgcat ttagtcaatt 35760 tatcagaaactaaaaatgtt gcaaatcccc atatgtgtga gtttcactat gctttttatt 35820 tccctgtaaagtatggtaag gtataaatga gtttatgaaa aatagaaaac aataattctg 35880 agtttagttttggatcttgg gttgcctggg catactcact agctaagtat ttttcacata 35940 ctagccatgaagtatgcatg attcatatcc ataccttagc aaaattgtaa accactatac 36000 tatctagtacttaggtcttt ttgtactcta ggatttgggg actcttaaga ttattctgga 36060 aaaaaaagtatagaagaaaa acagcaaaaa tacaccttca gtgccttatc ttagctatgg 36120 tcactgttatattgtcaagt attataaatt tgtattatgg tttttttttt tgagatggag 36180 tctcgctcacattgtgcagg ttggagtgca gtggcatgat ctcagctcac tgcaacctcc 36240 acctcctgggttcaagtgat tctccttcct cagcctccca agtagctggg attacaggcg 36300 tgcgccaccatgcctggata atttttgtat ttttagtaca gacgaggttt tgccatgttg 36360 gccaggctggtcttgaactc ctgacctcag gtgatccacc cgcctcagcc tcccaaagtg 36420 ctagggttacaggtgtgagc cactgcaccc agcctgtatt atggttttta aaaacatccc 36480 ctcttgttttcttcagataa aaatgataga aagacccaaa attgctgctg tctgtggaca 36540 ttatgattattattatgctc aacttgatat gctgaggagg agagcccaca aaccaagtta 36600 tcaccctattcctcaagaaa atactggagt tgaggattac ggtcaggaaa cgaggcatgg 36660 tccatccccaagtcaatggt aatattgtgg tctagcttaa gctttggtta atctaaaaat 36720 atctttatatattaacattt attattctga aatccaaatt ctcctaacac aaataatcca 36780 agaagaactttccaaatctt cattttaaac acatagttcc cttgaccttt ttcttttgtt 36840 tgcttttgtagacagtctca ctctgatgcc taggctagag tgcggtggcg caatctcagc 36900 tcactgcaacctctgccttc tgagttcaag cgattctcgt gcctcggcct ctccagtagc 36960 tgggactacaggcgtgcacc accatgccca actgattttt atatttttag taaagacagg 37020 gtttcaccatgttggccaac ctgatcttga actcctgacc tcaggtgatc tgcccgcctc 37080 agcctcccaaagtgctggga ttacaggcat gagccaccat gcctggccat gttagtccct 37140 tctttctatgtcagccctat acctgcttgt tagttggttc ttcaaattct caggtaccct 37200 ctcaccaggcagccactgac ctcatgtgat ccacctgcct tggcctccta aagtgctggg 37260 actacaggcaagagccacta ttcccagcct ttctttcttt tttttttgtt agaaagattt 37320 tgtttttatttccatcagaa tgtcatatat gttacacaaa tcaaatctgt tgacatctca 37380 agcttataacaattacgtgt tcttataaat tacgtgggaa ttacatgtac tgtgagaagt 37440 gttgtaattatgatgtaatg tatattataa tttagcctac agaagtaaca aagtcttgta 37500 attaaataaagcaataaatg tgttgataga ttattacaat tgataagtaa ttgataaatt 37560 atcttctttttcctgtaacc cttcttcatc tcaagtctga tctagcttat tttcttattc 37620 atagagctgcttaactgtag gcacagaccc atacccttgc tcttttaata ttctttcttc 37680 ctcctactaaattccactat atggcaggtg aaaaaatagt tgtgtatatt tcatttctca 37740 aagaggttactaatatgaat caataattga atcattaaaa tcaaatgatc atttgagaca 37800 ttttgagaaataagatatat ttcattcggc atttatgttc tagggatttt caaaatatgg 37860 acatgttagaaagaaaatag tattcttaaa ttggtcttat ggtagatttt caaaaaattt 37920 actccataatagatttctgc agatctacaa tattttcaaa tttttttcac actgatgttg 37980 acattcctgtgttcagaata attgacacct aacagaggcc tgaagactta agtctaagag 38040 ttctattttaaaaatgtttt gtcatcaatt ttttttgttc agggttaaga atttgttact 38100 ttggcgactctgattgttta ttgttgaaat tttggatgaa ttatgaaaaa cacaagatac 38160 tatgggatggcaatctcatt aatagtgaaa atgagataag caagaatgat aagagaataa 38220 tttcttcagaaacatataac gggaaaagca tatgttttat ctttaaagat attgggcact 38280 gttgtggtttttatcgatct tcataacaca tttttaatta tctccacaat tatcaaaagt 38340 tatgtcttctgtccgttcaa attgtaatat ccatattgga ctcaattagt gagggacata 38400 gattttacagaagaactgga gcagccacaa aactcccttt ctcttttctc aaccactggg 38460 aaaatatatcttccactctt tgtctagatt tgagagctgt caagctatca attattttga 38520 ccacatgtgattttatatct cccaagctct cacatgaaac agtaggaagg gtccttctct 38580 ttcctggatgcccctttgat gcctggtaac ccctcctctt tgtatactcc ctcatcccca 38640 gctttctgtctgctggaggt ccaattacag gccatgggat ggaggaaaag gatttttttt 38700 ttttttgagacagagtctcg ctctgtcgcc caggctggag tgcagtgacg cgatcttggc 38760 tcactgcaagctccgccccc tgggttcaca ccattctcct gcctcagtct ccagagtagc 38820 tgggactacaggcgcctgcc acgacacctg gttaattttt ttgtattttt agaagagaca 38880 gggtttcactgtgtcagcca ggagggtctc aatctcctga ccttgtgatc cacccgcctc 38940 ggcctcccaaagtgctggca ttacaggcat gagccaccat gcccggccga ggaagaggaa 39000 tttgtataggatttgggggg tggagaggga ataggtagac agagagatag agaatgtctt 39060 ttggacagcccctgggtgtt ggaatcattt ttctcatgaa gatattgata catgtgccag 39120 ttaggcttatgagacagatg agtgctacaa tttaccctca tttgattcaa gaactatctg 39180 tgtgtcaggcacttcacagc cctaatctct tctaatcctc gtatcaattc ctgtgaaatg 39240 gtaccatgcccactttacag ctgaggaact aagactcaaa gactttagct tgtcatttca 39300 tcctatttctaaatccctga ttattaactt gcctctttgt aaattgggga tgcttatcat 39360 gatgttccttcctaaaggag ttatttctga aattacagtt ctgtctttgg agccttagaa 39420 gttactcgtattccaaaaaa cttatggtct gaaatgcggt ttttatttag caaccaataa 39480 ttacagaaatgttttacagg aaattctgcc aaaaaaaaga tacataaaat gtgagtataa 39540 acttgaaaattgtttgactg gaattgacta aaattgtgct ggaaaaatac cttaaacatt 39600 tggagagacagctaaaccat tatttctttc ctcattaagc atttatgtgc ggagataaag 39660 ggatggatggagggacacat tctgctctca gggagctcag tatgtggtgc aggaaacaga 39720 tatgcagccattcttttttt tcttttcttt tctttttttc tttttttttt gagatggagt 39780 ctcactctgtcactcaggct ggagtgcagt ggtgcgattt tggcttactg caacctctgc 39840 ctccctgctcagcctcccca gtagctggga ttacaggtgc ccaccaccac gccccactaa 39900 ttttttttagcagagacggg gtttcaccat gttggccagg ctgatctcga actcctgacc 39960 tcgtgatccacccacctcgg cctcccaaag tgctggggtt acaggtgtga gccaccacat 40020 ctagccactattgttaaatc aggagcgaca acctgtacat tagacaccta cacaaagcgt 40080 gagaacttctgggtgtgggt ctgttttcct ccccacaaca tattatagag aatggaagga 40140 ctgaatcttgtcctgaagaa aaatcactgg ataagaatat ttttctgttt aatcctctcc 40200 tgtatccccacttgttactc ttcatccttt tttccttttg attccaaaat tttcttttcc 40260 aatgtaaagattctgtaact gtgaactact tcttgaactt ggaacttcaa gccactggtg 40320 aattgtgaatctcattacta aactgaaaat tactcgtcaa attggtgcct aagattcgtt 40380 caagtttctacttaagctga acattcttat tttctaaggc ctgctgagta ccttcagaga 40440 aaatttgaagctcaacaata taagttgaaa gtggagaagc aattggtaag taaaatacca 40500 aatatgggaagcaattagga atttcctaat agtttttctg ttcacagatt ttcaagtcaa 40560 agttcattccaccagaaggt caagaatact ctctactagt ccccagtttt ttttgttttt 40620 gtttttgttgttgttgttgt tgttgttttc tgagacagag tctcgctctg tcaccaggct 40680 ggagtgcagtggtgtgatct tggctcactg caacctctgc ctcccaggtt caagcaattc 40740 tccttccccagcctcctgag tagctgggat tacaggcgcc caccaccacg cccagctaac 40800 ttctgtatttttagtagaga cagggtttca ccatgttgtc caggctggtc tcgaactcct 40860 gatctcgggtgatccaccca cctaggcctc ccaaagtgct ggggttacag acgtgagcca 40920 ctgcacctggcccgagtccc cagtttttaa tagctaaata aaataatggg aacaggcttg 40980 aatcacccccttagcagtcc ggtttcttcc ttggctctat ctcttctgtg ggaccttgga 41040 cagttcattcagcctatctg agccttaatt tccttttcta taaatgacaa tttttagagt 41100 agatgagcttcaaatttcct tgcagtgctg tagtgctttg gttctatttt gttaaagatt 41160 ctgctgcacattaaaaaaag tgacaagggg ccaggtgcgg tggctcatgc ctgtaatccc 41220 agcactttgggaggccaagg tgggcggatc ataagatcag gagttcaaga tgagcctgac 41280 caacatggtgaaagcccgtc tctactaaaa atacaaaaat tagccaggca tgatggtgca 41340 cacctgtaatcccagctact tgggaggctg aggcaggaga attacttgaa cccaggagga 41400 ggaggttgcagtgagccgag atcgccctac tgcactctag cctgggcgac agaacgagac 41460 tctgtctcaaaaaaaacaaa aaaaacaaaa accaacaaca aaaaagtgat taggccagat 41520 attatggctcatgcctgtaa tcccagcact ttgggaggct gaggtgggtg gattgcttga 41580 gcccaggagttcgagactag cctaggcaac ataatgagac cttatctcta ccaaaaaaaa 41640 caaaaattacccaggtgttg tggtgtgtgc ctgtagtccc agctactgag ggggctgagg 41700 ccggaggattgcttaagctt gggaggcaaa ggttacagtg agctaagatt gcgccactgt 41760 actccagcctgggtgacaga gtgagactct gtcttaaaaa aaaaaaaaaa aagaaaggct 41820 gggcttgatggctcatgcct gtaatcccag cactttggga ggccaaggcg ggcagatcac 41880 gaggtcaggagattgagacc atcctggcta acacagtgaa accctgtctc tactgaaaat 41940 acaaaaaattagccgggtgt ggtggcgggt gcctgtagtc ccagctactc ggaaggctga 42000 gacaggacaattgcttgagc ctaggagttg gaggctgcag tgagccaaga tcatgccgct 42060 gtactccagcctgggtgaca gagtgagacg ctctcaaaca gaaaaaaata tatatttttt 42120 aatgctttataattaagaaa attctactac ttaccacaaa aaaaactccc aaatactgag 42180 tttgcttagtgatataattc ttatttatag gaaaaagtca atgtcaaatc agaagatgtt 42240 tccgaaatcaaagtatgcat tataaattat ttcattcaat aaatagggtc ttcgtccatc 42300 ttctgccgagccaaattaca accagagaca agagctaaga agtaatggag aagagcctag 42360 attccaggagctgccattta ggaaaaacga aatgaaggaa caggttaaaa actgtttaat 42420 tccagggctacccttgtatt tctttgtatt actgtctttt gtactgtaat agggagttac 42480 ttctatttcctacagtgccc ctgaatatgt caacaccatg ctgagtgtta taggggatac 42540 agagttaggtatttcacttt ctcagataat gcgatatggc taagttcata aagcttttca 42600 ccttgagattcatagagtaa ctgtccatca gtaacaggtt tgggatttgt attagtttcc 42660 tgggctgctgtaacagagtt caacaaacta ggtggcttaa cacaatagaa atgtattgtc 42720 tcacaattctggaagagtgg aaatagaaaa tcaaggtgtc agcaggacca agtgccctct 42780 gaaacctgtaggggaatcct tccttgcctc ttcctagctt ctggtgcgtc actggcaatc 42840 tttggcatttctttacttgc agtcgcatca ctccattctc tgccttcatc acatgctgtt 42900 cttcctgtgtgtccctgtct tcacatggcc atcttcttgc aaggacatca gtcatattga 42960 actaagggcccactggtatg atctcatctt aagtagtctc atctgcagcg accctgtttt 43020 caaatggggtcacattctaa ggcactgggg gttaggactt caacatatct ctttttgggg 43080 aggaacaaacttcagtgcat aagagggtta tatataaaag tgggatttat aaagtaagtg 43140 tacatcatgaacacatttgg gttatatata aaattgagct ctgtagctaa agccactgtc 43200 tcacagggagtgaagtactg cagccaaaac ataaggcaga ttatcatctt taggagcaac 43260 atatttttctaaccttattt tatattacac acttttgaaa ttgtaggctg cagaaagatt 43320 atttttgttatggtgttcat aaacatttaa agtttctgga ttgggtttgc tttccaggaa 43380 tattggaagcagttagagga aatacgccaa cagtaccaca atgacatgaa agaaattaga 43440 aagaagatggggagagaacc agaggtaaat tcattcttct aggggaaaca ttgttctatc 43500 gatttagagctaactaaatt gagctggtat taaaagtaat gatttcctta tagaaaagat 43560 aaagttttatcatagagata atcatgtaga cttctttttt aataggaaag ctgtcagacc 43620 tcattggagcttcagtttat tatggtttat gagagactac acaagataat aaggatatct 43680 gagattctcaggaatggcta ttattaaaag tacttattga ttgtttcctt catgaatcac 43740 tcaatacatatttattgagt ggcaacttta gacaagagct ggattagatg cagaaagtcc 43800 aaaatgagtgtaagttcatg cccagaaggt gggaaaaaaa caaacccaac acactagcat 43860 tttttcaactctctgcaggg taaggttcta aaggctattt aaggcaaaat ctgagtgcag 43920 ttgaactgattcttaaaaat ctcttaaagg cgccacattg gaaattcatc cttccatctc 43980 ccaagaaggtctctagagtt ggcacagatc actgcttctt cagaagagct tcacatgaaa 44040 tagccagcctgtgtttggaa accatgttgt aagaaagaca catggctatt gaaacactag 44100 gaacacactcagtgccctgg aatgctctcc taggagaagc ttgcaggcac tgagacagct 44160 gtctcccatcccacatgcac ttggccacac actcattgag tagagctacc atgctgctga 44220 aattgatctctctctctctt tctcccaccg cagtgcatac agataaattc atataagtca 44280 aatgaatgtatggtgcaatt cagttgtgtt tgccaggcca tgaactagag ctttcacata 44340 ctgtattagtctgctctcat actgctaata aagacatacc caagactggg taatttataa 44400 agaaaaagaggtgtaataga cgcacagttt cacatggctg gggaggcctc acaatcatgg 44460 cagaaggcaaaggaggagca aagtcatgcc ttacatgaag gcaggcaaga gagcttgtgt 44520 aggggaactcctatttacaa aaccatcaga tcttgtgaga cttactcact accattaaaa 44580 tagtatgggagaaaccaccc cgatgattca gttatctcca cctggcccca cccttgacaa 44640 atgaggattattacaattca aggtgagatt tgggtgggga tacagagcca aaccatatca 44700 cgttcatactttcttttatt tacccctgta cagagaagtt aagtagctca tccaaagtca 44760 cgtagctattacaaggcaga cgaaatattt aaatatctga ctctaggctg ggcacggtag 44820 ctcatgcctgtaatcccagc aatttgaaag gctgaggtgg gaggattgct tgagcctagg 44880 agtttgagaccagcctgggc aacataggga aaccctagct ctaaacacac acacacacac 44940 acacacacacacacacacac acacacacac acacacacac tctctctctc tctctctctc 45000 tctctcactctctctctctc tctctctctc tttaaattag ccggacatgg tggtttgcac 45060 ctgtagtcctagctacttgg gaggctaaag cagaaggatt gcttgagcta ggagctaaag 45120 gctgcagtgagccatgattg tgccactgta ccccagcctg ggatacagag caagactcgg 45180 tctcaaaaaaataaagtaaa ataaaatgaa aatctgactc taaaacccct actcatgttc 45240 atgcctgtaatcctagcatt ttgggaggcc aaggcagaag gatcgcttga gcccaggagt 45300 ttgagaccggcctgggcaac ataatgagac tccatatgta caaaaaattt aaaaaattag 45360 tgggtcatggtggcaaatgc ttgtagtccc agctactcag gaggctgagt tgggaggctg 45420 aggttgaagctgctgtgaac tgtgattttt ccactgcact ccagcctggg caacagaggg 45480 aggccctgtcccaaaaaata aaaaaataca attataacca ctattctttc tggcatatgc 45540 agttctacttataaatggtt ggaatgacga gacacatgta taaaacaatc atagagtaag 45600 ccctgtaggtgcacaggagc caagatgagc agaatgagca ggtagcgggt atttatggaa 45660 gaagtgggtggggcctaaag agtggaattt ggagaggcag agttaaagga ggagagtggg 45720 cattctgaaagaaccatgca aaggttggga cagaggactg tgtgtgctgg gaggggcagc 45780 ctggaggttgttctctctgg agcagaggcc tggctcatgg gcagcctgga gccatcatca 45840 gccttatgtctaaggctgat ctgggatggg cagcctgagc cctgcaaaaa tggatagcag 45900 acattggtcaggcgcggtag ctcacgcctg taatccccag cactttggga ggctgaggtg 45960 ggcggatcacgaggtcggga gatcgagatc atcctggcta acacagtgaa accctgtctc 46020 tactaaaaagccaaaaaatt agccaggcgt ggtggtgcgt gcctatagtc ccagctactt 46080 gggaggctaaggcaggagaa tggcatgaac tcaggaggca gagcttgcag tgagccaaga 46140 tcgcaccactgcactccagc ctgggtgaca gagtgagact ccatctcaaa aaaaaaaaaa 46200 tggtggatatcagacatttc tacagaggct atggaggaag gatttgggaa atactgaagc 46260 tgtggcggggaaaaggagct ataaaaaggg ttctgtccag ccatttcatc attgatcgcg 46320 catcagccaagtagccttca gagctcactc agaaccaatc ttgttgactg tgtataattt 46380 gtgttaaaggagaactcaaa aataagtcat aaaacctatt tggtgaagaa gagtaacctg 46440 cctgtccatcaagatgcatc tgagggagaa gcacctgtgc aggtaatgat ggctatgatc 46500 agatgtgtgtgcttgcagtg tgtgtgctct accccaagtg gctcttaccc ttctctgtgc 46560 agcagaaccacgtagggaac tttttttttt tttctatgag acggagtctc actctgtcgc 46620 caggttggagtgcagtcgtg cgatcttggc tcactgcagt gtctgcctcc tgggttcaag 46680 tgatttcctgcttcaggctc ccgagtagct gggactacag gcatgcgcca ccatgcccag 46740 ctaatttttgtatttttagt agagacagga tttcaccatg ttggccagga tggtctttat 46800 ctcttgacctcgtgatccac cagcctcggc ctcctaaaat gctgggatta taggcatgag 46860 ccaccgcctttttaccagaa cttttaaaaa ctcagatgcc tctctctgcc ccagatggtc 46920 tgggatgaggcccaggcatt ctgtctgcag aagcttgctg ggtgatttag taagcagcca 46980 agtttgagaactgctgctat ttagtataag aacgttccac tctctggagg gtctaagtca 47040 gtgtatcagacacattggtc aggaaatctg agtcaagttc tcttccattt caaccttatg 47100 tttttggtggaggtaagagc ctgggcagag ttgaaataac aaataaatct caagagagtt 47160 tttttcccttctgagaaaag ataatgcaat tataatacaa gatgaatctg ttgatttcaa 47220 ccaattctgagaattattaa acctgtgaaa tgacctgata aacaatgctt ttatggttac 47280 ataaaataattacataaaat gttttacttt ccaaggagtt atatttattt tgcgaataag 47340 aagcccaagtgtgcttttat ttttgcttag taagaagatt ctcaatgatt tggcccatac 47400 taagaattattattatcttt tttttttaga tggaatttcg ctcttgttgc ccaggctgga 47460 gtgcaatggcacgatcttgg ctcaccgcaa cctccgcctc ccaggttcaa gcgattctcc 47520 tgcctcagcctcctgagtag ctggaattat aggcgcctgc caccgcgccc agctaatttt 47580 tgtattttagtagagacagg gtctcaccat gttggccagg ctggtcttga actcctgacc 47640 tcgggtgatccacctgcctc agcctcccaa agtgctggga ttataggcat gagccacccc 47700 gcctgagcgaattattatta tctttataat tagagtaatt ctctgtgttt taaattatat 47760 ttattattagagcttggtcc agagtcaact agaaatggaa aatcctcaag gtattataaa 47820 cttgtcatttaaaggtgcca gtaggatcac agtcacattc cataaaaaca cggctcagat 47880 gttacagacatgtttttctc tcacattttt taacctggtt agagtaaatc cagtgcctta 47940 aagtttttaataagtcaggt aattaaaaat aaaccactgg aagcctcaaa aagtttgtat 48000 caggaattgggtgaataaaa tcttgtatat tttatgcaag aggagtaact ttgaaagaaa 48060 acacaccaaaatgccaatgg tggtaattgg tggtatctgg attggtgtga gtaggaatga 48120 ttattgtctctctacttttt agatttttta taagaaggtt acagaacttt tactacaaat 48180 atgtataataaagtatccgt tccttagttc tgtcagcact ctaatcaata tcttcaaaca 48240 aaaaagccatctgaaagaca gaaatggtgg cacgagacta tagttccagc tatttaggag 48300 gccgaggatcccttgagctc aggagtttga gaccagcctt ggtaatatag tgagacccca 48360 tctctaaaaaaaaagaaaag gcatctgata tttcctgaag gctcctccag agcaatccag 48420 cagcagatacctttgcaaac ttttgtaaag gaaataatta tcacttaatt tgtctaattt 48480 ttggatttaggttttaatta tcttttttga agggaatatg cagctatata ataagacact 48540 ttaaaaaagtctctacttgt agagttatct ttccaaaata ctgatttgaa cattatttct 48600 ctacacgacaatcaatggcg actgccattt ctcttagcat ggcatgctag acttttgtga 48660 gttgttcctaacagaatgtt ccagcctcat tgctcacatt tcccccaaac atacccaaag 48720 ctctaaatgtctcagattac cttttttttt tttaaatgac atatttttta tttctttaag 48780 tgatttttttcactgtggta aaatacatat aacatcgcct ttaccaccct aaccattttt 48840 ttttttttttttaattgatc attcttgggt gtttctcgca gaggggtatt tggcagggtc 48900 ataggacaacagtggaggga aggtcagcag acaaacaagt gaacaaaggt ctctggtttt 48960 cctaggcagaggaccctgcg gccttccgca gtgtttgtgt ccctgggtac ttgagattag 49020 ggagtggtgatgactcttaa cgagcatgct gccttcaagc atctgtttaa caaagcacat 49080 cttgcaccgcccttaatcca tttaaccctg agtggacaca gcacatgttt cagagggcac 49140 agggttgggggtaaggtcac agatcaannn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49200 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49260 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49320 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49380 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49440 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49500 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49560 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49620 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49680 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49740 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49800 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49860 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49920 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 49980 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50040 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50100 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50160 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50220 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50280 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50340 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50400 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 50460 nnnnnnnnnnnnnnnnnnnn nnnnnnnnnn nnnnnnnnnn ntacgaaaac cagtcaggcg 50520 tggcggtgcgcccctgcaat cgcaggcact cggcaggctg aggcaggaga atcaggcagg 50580 gaagttgcagtgagccgaga tggcagcagt acagtccagc ttcggctcgg catcagaggg 50640 agaccgtggaaagaggggag agggagaggg agagggagag ggagagggat cagattactt 50700 tttaaagccctacttattta aaaagacatc ttccttttaa cctccaggct tttgtaaaat 50760 gcttatttctctactgaaat atcccttccc tctcttctct tcttgcagaa cacatctatc 50820 agacctcctggtgaagtttc tagcacagct tttttctctt tctcccttag aattaataac 50880 tgcctcatctgtatttccac agcatttcca agtacttcat acaccagcct gtgtcagttt 50940 gaagcattattagctatttg ccctgcaaac ttgggaaggg tttttctgcc ttgcagtagt 51000 atgaagtctgaaatcaggac tatgacttat ctatcttact tatatttgtg aagttggttg 51060 tctgatttgcttggatagtc tggtcatctc aattgtacaa taagtgctcc attactttac 51120 tttccttaaaatacaacgat ctcagattcc aaccccaatc tactccagtg ggtgggacat 51180 tcaaccttagtgtgctgtca agctctccag ggtcatgtca tctgaaaggc cctcttggcc 51240 ctgtgaagactgattaactg tgtagccatg gagtctggga tcttgaggca ggaactctag 51300 gctggtgtgcagtctcttgc tcactactcc aatgtactgc cacagattag gacttgagtc 51360 cgccatctctttaaaaaaaa aaacagtttt attgagatat aattgatcat aataaaccac 51420 acatatttaatgtatatact ttataaaatt tggcaggcac acccatgaaa cccatcacca 51480 caatcaatatagtgaacata tccatcacct gcaaaagttt gttgcgccct tttgtaaact 51540 cctctctcttattctcccta cctctcctcc catctcatcc ccatgcaatc acggatctgc 51600 tttctgtcgctgtaggttgg tttgaatttt ctagatttgt ttggattaca taaatggagt 51660 cgtgctgtatgtgctctttt ttctggctac tttcattcac ataataatgt tgagatttat 51720 ctatgttgcacatattaata gttcattatt attctttatt gctgagtata ttctattgta 51780 tgaatgtatcaaaatttatt gatccattca ctgtagatgg atatttgggt tgtctccagc 51840 ttttggctattataaataaa gctgctagga acattcatat acaaatcttt tttttttttt 51900 ttgagacaagtttcgccctt gttgcccagg ctggagtgca atggcacaat ctcagttcac 51960 cacaacctctgcctgctgga ttcaagtgat tcttctgcct cagcctccca agtagctggg 52020 attacaggcatgcgccacca cgcctgacta attttgtatt tttaatagag acagggtttc 52080 accatgttggtcaggctggt ctcgaactcc cgacctgagg tgatccaccc acctcagcct 52140 cccaaagtgctgggattaca ggtgtgagcc actgtgcctg gctatcatgt acaaatcttt 52200 atgtggtcatgtgcttcttt tctttctttt gggtaaatac attggactgg gatggatgga 52260 tcatatagtaggtgtatatt taactttcag agaactacca aatggtgttc cagaatggtc 52320 gcactgtgttacactcccct tgacattgta tgagtgtttc agttctctct gtgcagctct 52380 ctcctctttgggtctttgtc tttcagactc tagcacctta atacccccca agccttgtct 52440 tatcaactcagggagttggc cacactcatc ttcggtttcc atccctgcac ctcttcagtt 52500 ccccatccccgcaccatggc ttgcaaactc tctcaagaca ggaggctggg gcagttgcag 52560 ggcttgtctcattggttttc tgtttcttag ggattactgt ctttcattgc tggatgtcta 52620 atgtattaaaaaccatttat ctattatatg tttgatttgg ctctttggtt gtttcaggtg 52680 cggaattaaatctggtttct gatactctgt cttggctgaa agcatacgtt ttcagtgccc 52740 actgctggagaggggtggag ggcactcaag agttccattt ggacattgag ttagagaagt 52800 tgtgagagtttacatacctg ctctggagcc tttaccccac tgttccctct gcatggaaaa 52860 tgctctccccagactggcat atgccaaggt ccaatatcat tccagggctt aaattgattg 52920 ccagataagctttgcctgta ttactctcac tccctactca ttttctgtcc tgttatccta 52980 ttttgttcccttgatagcac ttaacacttt ctgaaattat gtcattcgtt aactcattta 53040 ttacctatcctactccagta aaatgtaatt ttcgtgtcag cagggacctt tctggtcatg 53100 tccactgtgctaacccattt tgagggtttc tggcccctgg ggagtgctca gtgtgaattt 53160 gtggagtgaatattaagatg aagataatgc taagtaggca gttggatatg tgagtctgga 53220 gctcagaggagaggaaaagt gaagcctgaa gatacacatt taagagtctc tgcttaacag 53280 tggcatttaaatccatagga atgaatgaaa ccccttgtat tagggaatag aagagcagat 53340 ggcccaagataggatgctaa gaaacctccg aatatggagt tcacatctca gttgtgcctt 53400 tgaaattcttgtcatccact tttagttttc ttctcttcct acttgaaatt gcctaccaat 53460 tttcagagccctctccttcc tttataccgt catgagttgc gcactttgct tattttcctg 53520 attaagatcataagcctctt aagggaaaga tcctgtagtc aaaattacat tcttgaattg 53580 aattgggttggactggagtg gactggagtg ataagtattg tcacattata gaattccacc 53640 cactgaagtgcaagtgttaa atgtattaat atttcaagtt aatggatact ctgcccaagt 53700 ttttagttaattattattaa ctttccatta taaaagcttg tttttgttat taaatcaatc 53760 atcagatttaacgcagaaat caactcatgt aaacatacag tgagagaatt gtatttttct 53820 ctaaattttcaggacattga aaaagacttg aaacaaatga ggcttcagaa cacaaaggaa 53880 agtaaaaatccagaacagaa atataaagct aaggtaagaa atacttttgt ctttgggttc 53940 catattaaatagctggctgg ggagccacct tgtgatctcg gttgcctgca tgattttccc 54000 cctagtattttatagaattg ctctattttg tgatatgaga ccaatggttt taagaatcta 54060 taatgtcaaacaaaattgac ctagggagtt gtaattttaa ggcttttact gaattgctaa 54120 actttttttttttttttgct ttctcctaga agggggtaaa atttgaaatt aatttagaca 54180 aatgtatttctgatgaaaac atcctccaag aggaagaggt atgccattaa gtctaaattt 54240 ccattagtaggtatcagaaa atgcatatat cttaatagca tgtttcatga aattatttca 54300 caggctgtagggataatttt tttcaacttt tattttagat tcaggtggta catgtgcagg 54360 tttgttacctggatatgttg tgtgatgttg aggtttggga tatgaatgat cccgtcaccc 54420 aggtattgagcataataccc agtagttagt ttttcaagcc ttgcttccct cctttcttac 54480 ccccactgtagtagctccca gtatctattg ttgctatctt tatgtccatg agtacccaat 54540 gtttagctcccacttataag tgagaacatg cagaatttgg ttttctatcc ctatgtaatt 54600 ggttttctatccctatgtaa tttgcttagg atagtagcct ccagctgcat ccatgttgca 54660 tggacatgatttcattcttt tttatggctg catagtatcc catggtgtat atgtaccaca 54720 ttttctttatccagaccacc actgatgggc acctaggttg attccatgac tttgctattg 54780 tgaatagtgctgggatgaac atgtgagtat atgtgtcttt ttggtagaat ggtttgtttt 54840 cttttggatatatacccagt aatgggattg ctgggttgaa cagtagttct aagttctttg 54900 agaaatatccaaactgcttt ctacagtggt tgaactaatt tacattacat ttccgccaac 54960 actacataagcattcccttt tctctgcagc ctcgccaata tttgtttttt gactttttag 55020 taatagccattctgactcgt gtgagatggt gtctcattgt ggttttgatt tgtagttctc 55080 tgataattagtgatgatgag tattctttta tatatttgtt ggctgcttgt atgtcttctt 55140 ttgagaagtgtctctttcta tcttttgtcc actttaaaat ttgggttgtt ttttcttgtt 55200 cagttaagttccttatagag tctggatatt agacctttgt tggatgcata gtttgcaaat 55260 attttcttctattctgtagg ttgtctattt actctgttga tagtttcttt tgctgtgcag 55320 aagctccttagtttaattag gttccacttg tcaattttgt ttttgttgca attgcttttg 55380 aggacttaatcacaaattct ttcccaaggc ccatgttcat aatggtgttt cctaggtttt 55440 cttttaggattcttatagtt taaggtctta cttttaaatt gttaagtcat ctttagctga 55500 tttttgtatacagtgaaagg taggggtcca gtttcattct tctgcatgta gctaaccagc 55560 tatcccagcaccacttattg gataggaagt cctttcccca ttgcttattt ttgtcgattt 55620 tgtcaaagattatatggctg tagatgagtg gctttatttc tgggttctct attctgttcc 55680 ttggtttatgtgtttgtttt tgaaccagta ccatacagtt ttgattactg tagtcttatg 55740 gtatagtttgaagttgggta atgtgacgac tctggcgttg ttctttttgc ttagaattac 55800 tttggctatttgggctcttt tttgtttaca tatgaatttt agaatagttt tttttttctc 55860 caatcctgtgaaaagttaca ttggtagttt gacaggaata gtgttgaatc tatagattac 55920 tttgggcagtatggccattt taatgatatt gattattcca atccatgcat gtggcatgtt 55980 tttccatttgtttatgtcat gtatgatttc tttctgtgtt gtgtagctct tcttgtagag 56040 atctttcacctccttggtta gatgtactcc taggtatttt attttatttt ttggtggcta 56100 ttgtaaatgggattacgttc ttgatttggc tctctgcttg aatgttattg gtgtatagga 56160 atcctattgattattgtact tcgatattgt atcctgaaac tttgctgaag ttgttcatca 56220 gttccaggaacctttgggtc gagtctttgg gttttcaacc tatagtatca taagcgtgaa 56280 gagatggtttgacttcttct tttcttattt ggatgcctag aattttagaa aatatttcta 56340 gaaaaatgtttggtgctcaa ggccagggaa cggtggctca cagctgtaat cccagcactt 56400 tgggaggctgagacgggcag atcatgagat caggagattg agaccatcct ggctaacatg 56460 gtgaaaccccatctctacta aaaatacaaa aaattagctg ggtgtggtgt cacccacctg 56520 tagtctcagctacttaggag gctgaggcag gagaatcact tgaacccagg aggcagaggt 56580 tgcagtgagctgagatcgct gtactgcact cgagcctggg caacagagtg agacactgtc 56640 tcaaaaaaaaaaaaagggaa agaaaaatgt ttggtgttca aatgagtcct ccaaatactt 56700 tttattctcccattttattt tattggtgtt atttctttag ataaattatt acattttaat 56760 ttacttttctttaaataaaa gagctatttt actcataata ttaattttta tcatagccaa 56820 attaaaatagaagacctgat acattgtcaa caactaatat actgacctaa aaaattgaac 56880 aggtaccctgaaaccaggca catttatttt aggtcttaat tagttattga taactttaag 56940 taaatctcatttatgcattt gggctctcct tgccacagca aggagtaaat acagtaaatc 57000 caatacagtaaatccaaatt tcattttatt agttgatttc aaaatctttt tttatcctgg 57060 ttttatcagacctataacaa atgtcaaaat taattggttt atttttccat tttacctttt 57120 ctgaattcaccttttaagtc aatataagta tgaataatta tacctgatgc tcagttttta 57180 tttaatgttctttattagct taaaacattt tcatgttagc atttcttatt tttatgagca 57240 tttgctacataaagacttca ttagagtggt gatagttagc attcacctct gttcaaccat 57300 aaattcctaaatgccccaga ggtgagacat cagagtggag cagatctggg gacctgcttc 57360 tgagtgggaacttgagaagt ggtactctca cagagcttct gtgaagtgag gtgctgacgt 57420 tgccctgctgaaatgaaaga atggagtcca aaaagtttta actgccactc tttcttattc 57480 tttgctttgatctgcgtgaa acagaagtgt tcattttggt attgactaca aaatactagg 57540 agcagatttagggagctggt taagaatgtt gtacacttaa aacagggcat gaatgagaaa 57600 agcttgagagcactgtagaa tggagctgaa gtggaatact attgaagtca gaaagtctag 57660 ataaaattaagttgccttat gaccagtgct tgacactgtt aacatggaga agaaatgaaa 57720 acatttctgtttttatctaa catagctcta gttttaaaac tctatggatt tatttgttta 57780 gtaaacatttgttgaatatt tactatatac cttgctaatt aattttacta ggaacacgaa 57840 aatatggtttttctttcttt caaaatatgg ctaatttatc atgaaacact gtggaattga 57900 tttaggcaatggatatacca aatgaaactt tgacctttga ggatggcatg aagtttaagg 57960 aatatgaatgtgtaaaggag catggagatt atacagacaa agcatttgaa aaacttcact 58020 gcccagaagcaggtatgtgt ttcttgaaag ttgtaaatga gaaggaactg ttttattagc 58080 aacccattttgaactctgtc cccatgcatc tgcctcggct ccactgttac ttgacccctt 58140 tctgccctctctaagcaagg cagaaacaca cttattattc tcctgccacc catgcagtgg 58200 ccacactccctgagatccag ccctcctctc ctgctccata cccactccct cttgcagctt 58260 tggcttctcccaggagctcc agacttacca gtctttctca ttgtcttctg ggaagctcca 58320 tggacaagtgttgccagtat ctgaaactca gctgtgtaaa gtcaagctct tctgtgctct 58380 tcccagtgaccctttatttt ggttagtgtc acagatgcaa ctggctgggg ccagtgttgt 58440 gggcagtaaaagaatttatc aacacaattg taagtaaaga aaggcagatt tattaaagta 58500 cagagatacgttgcaagagt gcaatgggca gcacagcaga gaagaggctg tctgctaaga 58560 ggcaggggctagagggaagt tttatagggt catattggag gagctacatg ctgataaggt 58620 gtgcagataaggttttgctg cttgggctac atgtggaagg aatgaggtat ttgggaacag 58680 gatgtgacagcagcttgtct gtgatgagtc atctctcaga acagttgttc ccccatcccc 58740 acccccaacctgggacccct ccctttttgt tgtttactta tcttatgaga acttcacagt 58800 cagtgctgtcaccagggtgc acccttagca tagtgtctat tctgagatgt ctctggagtc 58860 ttcctcttccttcattctct ctgttactgg tttagggctc tgtcatctct cagtagtgtg 58920 gtgtaggcttcagagacaga tgggaattga atctcagctg ttgctgccac cttctggtta 58980 tgtgacctttctttcacaag ttattccaac actgaatctc agtttcacct taggaacagg 59040 ggataatagtagtaggaata accacacagg gtaattgtga ggaccaaagt gagttttgat 59100 gtataaacgacctggcacat actaggtgcc taaattaagt gctgtctttt cattttccct 59160 tttccttccccttgctgtat tgccttattt gcttatgtga ccttctttct ctagtatttc 59220 cccttcattctctaaatggt tactgtatta gtccattttc ttgctgctga taaagacata 59280 cctgagactgagcaatttac aaaagaaaga ggtttaattg gacttacagt tccacatggc 59340 tggggaagcctcacaatcat ggtggaaggc aaggaaaagc aagtcacatt ttacatggat 59400 ggcagcaggcaaagagaaag aacttgtgca gaggaactcc tctttttaaa accatcttat 59460 ctcgtgagactcattcacca tcacgagaac agcatgggaa agatccgccc ccatgattca 59520 accacctccctctgggtcac cccacaacac acaggaattc aagatgagat ttgggtggga 59580 cacagccaaacgatatcagt tactaaagtt atcttggcat attattactc tgctcagatt 59640 ttttttttggataatacctg cagaataagg tccattccac atattatcac atttaacact 59700 acatggcctaattctgctgt gacccacttt tctcatccca gcatggcctc tttccttcca 59760 tggaaaatgggatccataca gcctgctgga atgcccattt tctcctacag ctggaatgcc 59820 cattttctcctacagcattt acagaactga cttggctcag tttcctcttc ctggaatact 59880 ctctgcctcatttccttctg gaaaaatctc cattcagcag gcatcttatt gaggatctcc 59940 tttgtgccaaagactgctca ctggtaggga gctcaaagat gaatgaaatc tgggccctgt 60000 tctcaatatcacagaagtgt tatgagcaaa aaagtcacaa aacatgtttt ctgagcctga 60060 aatgttaatcactgtttgaa gtgcgagctg ggtggagagt cagggaggtc cgcactcctc 60120 cagggcttcacatgccatca tttttgtgat tgagaaggat catgctggct gcagagcaaa 60180 ggatggcatggagggcaaga ctgaaggcag gagaagagtc caagtgcatg agccagagtg 60240 gtgcagggagaatagatact gagtgtggga actgaggaag agaaggggct caaggatatt 60300 cccagttttctaattcaaat gcatgaagct ttcatcaacc aaaaatacat cacatggagg 60360 gtaatggggtcgggagagac aaggtagtga tctaaatttg gaacatgttg agatttaggt 60420 ctatagagcatcagttgcag attctatata agactgaagg cctggggcat atcagggata 60480 aagatatagcttggtggccc ttagcatatc cgtggttttt aactttggtg atggtcaaaa 60540 tacctatgcagaaggactgg agtgagaagg aaatggagct taggacataa ccctaccact 60600 atataaacaaactttggaga atcaggagag agtaaagcca aaggaggaga gacaggtcat 60660 ggaggaggcacaggaattgg cagcatcaac tggaagagaa aggccagatg aggtgagtgg 60720 gatttggcccttcaggagcc gttaatggcc tcagggaaag cagtcaactg tgtaaggggt 60780 aaattcaatggttatctttg catcagtttg ctgggaaaag cagagggggt tggctgtttt 60840 ttagatgaaagaaaaaaaaa ccttcatcag tagtatactg aaaattgtct ctcattttaa 60900 tctgtattcctgtaattatt atttaggctg aaggattttt ccgtatgttt gttgaccatt 60960 catatttctccttttttttt cttttttctt tttttgtttt ttttttgaga gggagtctcc 61020 ctctgtcgcccaggctggag tgcagtggca caatcttggc tcactgtaac ctccgcctcc 61080 tgggttcaagcgattctcct gcctcagcct ccctgagtag ctggcatagg tgcgcgccac 61140 cacgcctagctgatttttaa aatattttta gtagagatga ggtttcacca tgtttgccag 61200 gctggtatttgaactcttga tctcaggtga tctgcccacc ttggcctccc aaagtgctgg 61260 gattacaggcatgagccacc acgcctggcc aacccttcat atttctgtta tgaattatgt 61320 actcatgcccttcatccttt tttctactga aaatggcatg tttgtttttt tctttataag 61380 actgatttaaatcaaacctt tgcctgtaat atgtattgca aatgttttcc tcagttggtt 61440 gtcagatctcatttatagta ataacagcaa atatatatga gtgtgtgtgt gtgtgtgtgt 61500 gtgtgtgtgtgtgtatttgt gtattcatcc acttaggaat aaattttatg agaattgtgc 61560 ggcatatagaaagaaaactg taaaacctta ctgaggtatt tacagaccac ttgaataaat 61620 ggagagaaataacggtgcta tatattggaa atattttttc caaataaata ttgcagtatc 61680 gttgtctgaggtattaccca gaactctttg tctcacgacc aaaagaataa ggagggtgga 61740 cagtaagggtgagtttggac cgaaaattta ataaacaaaa gaggaaagct cttcactgtg 61800 gagaggggacccaagagggt tgccatttca cagctgagta caaaggcttt tatgaggaac 61860 ctgatagggctgggggtttc atttgcataa ggcatgaatt tctggcagct ccaccctgtt 61920 ctcctagtatgctgactggc taggggttgt ttttggaaaa ggcaccactc agaaaatgac 61980 atgatggttgaccaggcatg gtagttcatg cctgtaatcc cagcactttg ggaggctgag 62040 gtgggcagatctctcaaggc caggagttcg agactagcct ggccaatatg gctaaagccc 62100 atctctactaaaaatacaaa aattagccag gtgtggtggt gcacacctgt aatctcagct 62160 acttgggaggctgagccaca agaatcactt ggacctggga ggtgaaggtt gcagtgagac 62220 gagattgtgccaccacactc cagtcacact ccagctgggt gacagagcaa gcaagactcc 62280 atctcaaaaaaaaaaaaaaa aaaaatgacg tggtgtaaag accagttgga gccttggccc 62340 acaaccagctgagtgttgga gtgatggttc acagaggctt ggctcacagt ccaaagtatg 62400 ccccaaaaaggaaaggaatg tgctcactgg ggcccaccat gtacatgccc acaaaaggag 62460 aaggaactatttgctagagg cccactgatt gcacaaagaa caaaggcatt tctgtgttgg 62520 actttgctcccttatctgtg cagctgtggg catgttttag gcaagcttcc tgtgctagtt 62580 cccttatctgtgtctgcagc ttgatttttc agactgttct tttgtttgaa agaattctga 62640 ggacctgccctaactgcctg cctaactgat tctttctttc tcctccctca atatgtggat 62700 ttatggctatttcaatcaaa accacagtag gatttttttt ttaatggtat agggagatct 62760 tggcaggttggagaatcctg gagcttctta agtggccaaa aattttgaaa aagaagaaca 62820 gtgaagtggtacttacattt ccaaatgtca aaatatatta cagaaattat agtcattcac 62880 acaatatgatagtagcaccc aaatagttaa aacagtgaga agagaaagtt agaaacagat 62940 cctagtatgtatcataattc agcacaaatg aaaagtaaca tcacaagtca gcgtgaaaag 63000 aaaggattattcagataaat gctgctgggc caattggtta acagtttggg gaagattgtg 63060 aaatcagaccctatataata tgatacaaca aaataaattt tttaaaaaag agttatatgt 63120 aaaaagttatacattagaaa atgaaataaa agaacatagg tcattttttt ttttttttga 63180 gacagcgtctcactctgtca ccaaggctgg agtgcaaagg cgtgatctcg gctcactgca 63240 aactccgccttctgggttca agcgattctc ctgcctcagc ctcccgagta gctgggacta 63300 caggcacccgctaccacgcc cagctaattt ttatattttt gatagagacg gggtttcacc 63360 atgttggccaggatggtttc gatctcttga ccttgtgatc cgcccgcctc ggcctcccaa 63420 agtgctgagattacaggcgt gagccactgc acccggccga gttaattttt tttgaacagg 63480 gaagagctatctgttcaaaa tacatagaaa aaaaaaccac agaataaatt agtaataatt 63540 caactttaacaacaaaaagc tgtaataaag caaatcatac taacccct 63588 4 616 PRT Mouse 4 MetGlu Lys Tyr Val Arg Leu Gln Lys Ile Gly Glu Gly Ser Phe Gly 1 5 10 15Lys Ala Val Leu Val Lys Ser Thr Glu Asp Gly Arg His Tyr Val Ile 20 25 30Lys Glu Ile Asn Ile Ser Arg Met Ser Asp Lys Glu Arg Gln Glu Ser 35 40 45Arg Arg Glu Val Ala Val Leu Ala Asn Met Lys His Pro Asn Ile Val 50 55 60Gln Tyr Lys Glu Ser Phe Glu Glu Asn Gly Ser Leu Tyr Ile Val Met 65 70 7580 Asp Tyr Cys Glu Gly Gly Asp Leu Phe Lys Arg Ile Asn Ala Gln Lys 85 9095 Gly Ala Leu Phe Gln Glu Asp Gln Ile Leu Asp Trp Phe Val Gln Ile 100105 110 Cys Leu Ala Leu Lys His Val His Asp Arg Lys Ile Leu His Arg Asp115 120 125 Ile Lys Ser Gln Asn Ile Phe Leu Thr Lys Asp Gly Thr Val GlnLeu 130 135 140 Gly Asp Phe Gly Ile Ala Arg Val Leu Asn Ser Thr Val GluLeu Ala 145 150 155 160 Arg Thr Cys Ile Gly Thr Pro Tyr Tyr Leu Ser ProGlu Ile Cys Glu 165 170 175 Asn Lys Pro Tyr Asn Asn Lys Ser Asp Ile TrpAla Leu Gly Cys Val 180 185 190 Leu Tyr Glu Leu Cys Thr Leu Lys His AlaPhe Glu Ala Gly Asn Met 195 200 205 Lys Asn Leu Val Leu Lys Ile Ile SerGly Ser Phe Pro Pro Val Ser 210 215 220 Pro His Tyr Ser Tyr Asp Leu ArgSer Leu Leu Ser Gln Leu Phe Lys 225 230 235 240 Arg Asn Pro Arg Asp ArgPro Ser Val Asn Ser Ile Leu Glu Lys Gly 245 250 255 Phe Ile Ala Lys ArgIle Glu Lys Phe Leu Ser Pro Gln Leu Ile Ala 260 265 270 Glu Glu Phe CysLeu Lys Thr Leu Ser Lys Phe Gly Pro Gln Pro Leu 275 280 285 Pro Gly LysArg Pro Ala Ser Gly Gln Gly Val Ser Ser Phe Val Pro 290 295 300 Ala GlnLys Ile Thr Lys Pro Ala Ala Lys Tyr Gly Val Pro Leu Thr 305 310 315 320Tyr Lys Lys Tyr Gly Asp Lys Lys Leu Leu Glu Lys Lys Pro Pro Pro 325 330335 Lys His Lys Gln Ala His Gln Ile Pro Val Lys Lys Met Asn Ser Gly 340345 350 Glu Glu Arg Lys Lys Met Ser Glu Glu Ala Ala Lys Lys Arg Arg Leu355 360 365 Glu Phe Ile Glu Lys Glu Lys Lys Gln Lys Asp Gln Ile Arg PheLeu 370 375 380 Lys Ala Glu Gln Met Lys Arg Gln Glu Lys Gln Arg Leu GluArg Ile 385 390 395 400 Asn Arg Ala Arg Glu Gln Gly Trp Arg Asn Val LeuArg Ala Gly Gly 405 410 415 Ser Gly Glu Val Lys Ala Ser Phe Phe Gly IleGly Gly Ala Val Ser 420 425 430 Pro Ser Pro Cys Ser Pro Arg Gly Gln TyrGlu His Tyr His Ala Ile 435 440 445 Phe Asp Gln Met Gln Arg Leu Arg AlaGlu Asp Asn Glu Ala Arg Trp 450 455 460 Lys Gly Gly Ile Tyr Gly Arg TrpLeu Pro Glu Arg Gln Lys Gly His 465 470 475 480 Leu Ala Val Glu Arg AlaAsn Gln Val Glu Glu Phe Leu Gln Arg Lys 485 490 495 Arg Glu Ala Met GlnAsn Lys Ala Arg Ala Glu Gly His Val Val Tyr 500 505 510 Leu Ala Arg LeuArg Gln Ile Arg Leu Gln Asn Phe Asn Glu Arg Gln 515 520 525 Gln Ile LysAla Lys Leu Arg Gly Glu Asn Lys Glu Ala Asp Gly Thr 530 535 540 Lys GlyGln Glu Ala Thr Glu Glu Thr Asp Met Arg Leu Lys Lys Met 545 550 555 560Glu Ser Leu Lys Ala Gln Thr Asn Ala Arg Ala Ala Val Leu Lys Glu 565 570575 Gln Leu Glu Arg Lys Arg Lys Glu Ala Tyr Glu Arg Glu Lys Lys Val 580585 590 Trp Glu Glu His Leu Val Ala Arg Val Lys Ser Ser Asp Val Pro Leu595 600 605 Pro Leu Glu Leu Leu Glu Thr Gly 610 615

That which is claimed is:
 1. An isolated peptide consisting of an aminoacid sequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 2. An isolated peptide comprising an amino acidsequence selected from the group consisting of: (a) an amino acidsequence shown in SEQ ID NO:2; (b) an amino acid sequence of an allelicvariant of an amino acid sequence shown in SEQ ID NO:2, wherein saidallelic variant is encoded by a nucleic acid molecule that hybridizesunder stringent conditions to the opposite strand of a nucleic acidmolecule shown in SEQ ID NOS:1 or 3; (c) an amino acid sequence of anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidortholog is encoded by a nucleic acid molecule that hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or 3; and (d) a fragment of an amino acid sequenceshown in SEQ ID NO:2, wherein said fragment comprises at least 10contiguous amino acids.
 3. An isolated antibody that selectively bindsto a peptide of claim
 2. 4. An isolated nucleic acid molecule consistingof a nucleotide sequence selected from the group consisting of: (a) anucleotide sequence that encodes an amino acid sequence shown in SEQ IDNO:2; (b) a nucleotide sequence that encodes of an allelic variant of anamino acid sequence shown in SEQ ID NO:2, wherein said nucleotidesequence hybridizes under stringent conditions to the opposite strand ofa nucleic acid molecule shown in SEQ ID NOS:1 or3; (c) a nucleotidesequence that encodes an ortholog of an amino acid sequence shown in SEQID NO:2, wherein said nucleotide sequence hybridizes under stringentconditions to the opposite strand of a nucleic acid molecule shown inSEQ ID NOS:1 or 3; (d) a nucleotide sequence that encodes a fragment ofan amino acid sequence shown in SEQ ID NO:2, wherein said fragmentcomprises at least 10 contiguous amino acids; and (e) a nucleotidesequence that is the complement of a nucleotide sequence of (a)-(d). 5.An isolated nucleic acid molecule comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequence thatencodes an amino acid sequence shown in SEQ ID NO:2; (b) a nucleotidesequence that encodes of an allelic variant of an amino acid sequenceshown in SEQ ID NO:2, wherein said nucleotide sequence hybridizes understringent conditions to the opposite strand of a nucleic acid moleculeshown in SEQ ID NOS:1 or3; (c) a nucleotide sequence that encodes anortholog of an amino acid sequence shown in SEQ ID NO:2, wherein saidnucleotide sequence hybridizes under stringent conditions to theopposite strand of a nucleic acid molecule shown in SEQ ID NOS:1 or 3;(d) a nucleotide sequence that encodes a fragment of an amino acidsequence shown in SEQ ID NO:2, wherein said fragment comprises at least10 contiguous amino acids; and (e) a nucleotide sequence that is thecomplement of a nucleotide sequence of (a)-(d).
 6. A gene chipcomprising a nucleic acid molecule of claim
 5. 7. A transgenic non-humananimal comprising a nucleic acid molecule of claim
 5. 8. A nucleic acidvector comprising a nucleic acid molecule of claim
 5. 9. A host cellcontaining the vector of claim
 8. 10. A method for producing any of thepeptides of claim 1 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 11. A method for producing anyof the peptides of claim 2 comprising introducing a nucleotide sequenceencoding any of the amino acid sequences in (a)-(d) into a host cell,and culturing the host cell under conditions in which the peptides areexpressed from the nucleotide sequence.
 12. A method for detecting thepresence of any of the peptides of claim 2 in a sample, said methodcomprising contacting said sample with a detection agent thatspecifically allows detection of the presence of the peptide in thesample and then detecting the presence of the peptide.
 13. A method fordetecting the presence of a nucleic acid molecule of claim 5 in asample, said method comprising contacting the sample with anoligonucleotide that hybridizes to said nucleic acid molecule understringent conditions and determining whether the oligonucleotide bindsto said nucleic acid molecule in the sample.
 14. A method foridentifying a modulator of a peptide of claim 2, said method comprisingcontacting said peptide with an agent and determining if said agent hasmodulated the function or activity of said peptide.
 15. The method ofclaim 14, wherein said agent is administered to a host cell comprisingan expression vector that expresses said peptide.
 16. A method foridentifying an agent that binds to any of the peptides of claim 2, saidmethod comprising contacting the peptide with an agent and assaying thecontacted mixture to determine whether a complex is formed with theagent bound to the peptide.
 17. A pharmaceutical composition comprisingan agent identified by the method of claim 16 and a pharmaceuticallyacceptable carrier therefor.
 18. A method for treating a disease orcondition mediated by a human kinase protein, said method comprisingadministering to a patient a pharmaceutically effective amount of anagent identified by the method of claim
 16. 19. A method for identifyinga modulator of the expression of a peptide of claim 2, said methodcomprising contacting a cell expressing said peptide with an agent, anddetermining if said agent has modulated the expression of said peptide.20. An isolated human kinase peptide having an amino acid sequence thatshares at least 70% homology with an amino acid sequence shown in SEQ IDNO:2.
 21. A peptide according to claim 20 that shares at least 90percent homology with an amino acid sequence shown in SEQ ID NO:2. 22.An isolated nucleic acid molecule encoding a human kinase peptide, saidnucleic acid molecule sharing at least 80 percent homology with anucleic acid molecule shown in SEQ ID NOS:1 or
 3. 23. A nucleic acidmolecule according to claim 22 that shares at least 90 percent homologywith a nucleic acid molecule shown in SEQ ID NOS:1 or 3.